Process for transforming monocotyledonous plants

ABSTRACT

A process for the stable integration of a DNA, comprising a gene that is function in a cell of a cereal plant, wherein said DNA is integrated into the nuclear genome of said cereal plant, said process comprising the steps of: 
     (a) providing a compact embryogenic callus of said cereal plant; 
     (b) wounding said compact embryogenic callus or treating said compact embryogenic callus with a cell wall degrading enzyme for a period of time so as not to cause a complete disruption of tissues, and transferring said DNA into the nuclear genome of a cell in said compact embryogenic callus by means of Agrobacterium-mediated transformation to generate a transformed cell; and 
     (c) regenerating a transformed cereal plant from said transformed cell.

This application is a divisional of application Ser. No. 08/475,975,filed Jun. 7, 1995, which is a Continuation of Ser. No. 08/064,121,filed Jun. 23, 1993, now U.S. Pat. No. 5,641,664 which is a 371 ofPCT/EP91/02198, filed Nov. 21, 1991.

This invention relates to a rapid and efficient method for transformingmonocotyledonous plants generally, especially gramineous plants,particularly corn and other major cereals. The invention particularlyrelates to the use of either intact tissue capable of forming compactembryogenic callus or compact embryogenic callus obtained from suchtissue to obtain transgenic monocotyledonous plants.

This invention also relates to novel transgenic gramineous plants,particularly cereals, which can be obtained by the transformation methodof this invention.

BACKGROUND OF THE INVENTION

In recent years, there has been a tremendous expansion of thecapabilities for the genetic engineering of plants. Many transgenicdicotyledonous plant species have been obtained. However, many speciesof plants, especially those belonging to the Monocotyledonae andparticularly the Gramineae, including economically important speciessuch as corn, wheat and rice, have proved to be very recalcitrant tostable genetic transformation.

Difficulties have been encountered in achieving both: a) integrativetransformation of monocot plant cells with DNA (i.e., the stableinsertion of DNA into the nuclear genome of the monocot plant cells) andb) regeneration from transformed cells of phenotypically normal monocotplants, such as phenotypically normal, fertile adult monocot plants. Ithas been suggested that such difficulties have been predominantly due tothe nonavailability of monocot cells that are competent with respectto: 1) DNA uptake, 2) integrative transformation with the taken-up DNA,and 3) regeneration of phenotypically normal, monocot plants from thetransformed cells (Potrykus (1990) Bio/Technology 9:535). In general,direct gene transfer into protoplasts (using polyethyleneglycoltreatment and/or electroporation) has seemed to have the best potentialfor success. Protoplasts for use in such direct gene transfer methodshave most often been obtained from embryogenic cell suspension cultures(Lazzeri and Lorz (1988) Advances in Cell Culture, Vol.6, Academicpress, p. 291; Ozias-Akins and Lorz (1984) Trends in Biotechnology2:119). However, the success of such methods has been limited due to thefact that regeneration of phenotypically normal plants from protoplastshas been difficult to achieve for most genotypes.

Recently, success has been reported in the transformation of, andregeneration of phenotypically normal plants from, certain lines of rice(Shimamoto et al (1989) Nature 338:274; Datta et al (1990)Bio/Technology 8:736; and Hayashimoto et al (1990) Plant Physiol.93:857) and corn (Gordon-Kamm et al (1990) Bio/Technology 2:603; Frommet al (1990) Bio/Technology 8:833; Gould et al (1991) Plant Physiology95:426; and PCT publications WO91/02071 and WO89/12102). However, it isnot clear from such reports that their processes of transformation andregeneration are applicable to monocots generally, particularlygramineous plants, quite particularly cereals.

SUMMARY OF THE INVENTION

This invention provides a novel method for efficiently and reproduciblytransforming the genome of a monocotyledonous plant, particularly agramineous plant such as a major cereal (e.g., corn, wheat, rice, rye,etc). This method comprises the transformation with DNA of cells ofeither: a) an intact tissue of the monocotyledonous plant, which tissueis capable of forming compact embryogenic callus or b) a compactembryogenic callus, particularly its embryogenic sectors, obtained fromsuch intact tissue, such cells being competent with respect to: 1)uptake of the DNA, 2) integrative transformation of the plant genome,preferably its nuclear genome, with the DNA and 3) regeneration of thephenotypically normal plant (e.g., phenotypically normal, fertile adultplant) from the cells following the transformation of their genome. Suchcompetent cells are preferably obtained by wounding and/or degrading theintact tissue or the compact embryogenic callus of the plant, forexample by: a) cutting either the intact tissue and the cells thereof orthe compact embryogenic callus and the cells thereof obtained from suchintact tissue; and/or b) depending upon the nature of the intact tissueor the compact embryogenic callus, treating the intact tissue or thecompact embryogenic callus with an enzyme to degrade the cell walls ofthe intact tissue or compact embryogenic callus.

The resulting wounded and/or degraded, intact tissue or compactembryogenic callus, containing the competent cells of this invention,can be transformed, preferably by direct gene transfer, such as by meansof electroporation, with one or more DNA fragments (e.g., foreign DNAfragments), preferably linear DNA fragments. Preferably, at least one ofthe DNA fragments contains a gene which can serve as a selectable or ascreenable marker, preferably a selectable marker, for transformed plantcells. Such a marker DNA fragment can be located on the same DNAfragment or on a separate DNA fragment as another gene or other gene(s)of interest.

The transformed cells can be separated in a conventional manner fromnon-transformed cells by culturing on a selective medium, preferably fora prolonged time, and the transformed cells, thus selected, can beregenerated in a conventional manner into phenotypically normal plants(e.g., mature plants) which possess the gene(s) of interest stablyintegrated in their genomes, particularly their nuclear genomes.

This invention also provides: novel competent cells of monocot plants,especially gramineous plants, particularly cereal plants, the genomes ofwhich have been stably transformed with one or more DNA fragments; cellcultures consisting of such transformed cells; phenotypically normalplants (e.g., phenotypically normal, fertile plants) regenerated fromsuch transformed cells; and seeds of such transformed plants. Among suchtransformed cells, cell cultures, plants and seeds are those transformedwith a DNA fragment containing a gene that encodes a protein capable ofkilling or disabling a plant cell in which the protein is expressed andthat is under the control of the tapetum-specific PTA29 promoter wherebythe plants are male sterile. The transformed gramineous plants of thisinvention, particularly transformed corn and rice, are characterized bytheir being from plant lines, from which it is practically impossiblewith conventional techniques to regenerate the transformed plants, asphenotypically normal plants, from transformed embryogenic suspensioncultures or from transformed protoplasts, particularly where for every10,000 untransformed protoplasts of such plant lines, no more than about500, especially no more than about 100, particularly no more than about10, quite particularly no more than about 1, phenotypically normalplant(s) can be regenerated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: NPTII gel assays of Example 2 of five corn transformantsobtained by electroporation of immature zygotic embryos.

FIG. 2: Southern blots of Example 2 of genomic DNA of one of the corntransformants of Example 1 (H99-M148-1), using the sequence listed asSeq. Id. 2 as a probe. Lengths of standard fragments are indicated. Theorigin is indicated by O.

Lanes:

1: PstI digested DNA of phage lambda+HindIII digested pTTM1 (positivecontrol--probe should hybridizes to 2824 bp pTTM1 fragment)

2: BglII digested genomic DNA

3: EcoRI digested genomic DNA

4: EcoRV digested genomic DNA

5: HindIII digested genomic DNA

6: BamHI digested genomic DNA

7: PvuI digested genomic DNA

8: PvuII digested genomic DNA

9: PstI digested genomic DNA

10: EcoRI digested plant genomic DNA of untransformed H99 plant(negative control)

FIG. 3: NptII gel assays of Example 4 of seven transformants obtained byelectroporation of compact embryogenic callus fragments derived fromimmature zygotic embryos.

FIG. 4: Southern blots of Example 4 of genomic DNA of one of the corntransformants of Example 3 (Pa91-M146-2) using the sequence listed asSeq. Id. 2 as a probe. Lengths of standard fragments are indicated. Theorigin is indicated by O.

Lanes:

1: PstI digested DNA of phage lambda+HindIII digested pTTM1 (positivecontrol--probe should hybridizes to 2824 bp pTTM1 fragment)

2: BglII digested genomic DNA

3: EcoRI digested genomic DNA

4: EcoRV digested genomic DNA

5: HindIII digested genomic DNA

6: BamHI digested genomic DNA

7: PvuI digested genomic DNA

8: PvuII digested genomic DNA

9: PstI digested genomic DNA

10: EcoRI digested plant genomic DNA (negative control)

SEQUENCE LISTING

Seq. Id. No. 1: sequence of pDE108

Seq. Id. No. 2: sequence of probe used to detect chimaeric neo gene inSouthern hybridizations

Seq. Id. No. 3: sequence of a DNA fragment of plasmid pTTM8 used in theconstruction of plasmids pVE107 and pVE108 and comprising the promoterfrom the TA29 gene of tobacco and the barnase gene

Seq. Id. No. 4: sequence of pDE110

DETAILED DESCRIPTION OF THE INVENTION

In monocots, embryogenic callus can be of two distinct and well knowntypes (see: Vasil (1988) Bio/Technology 6:397; Armstrong and Green(1988) Crop Sci. 28:363). One type of embryogenic callus can best bedescribed as compact and/or nodular and can often be considered asorganized. Such callus, termed herein "compact embryogenic callus", isused in accordance with this invention. The other and generally lessfrequently occurring type of embryogenic callus can best be described assoft, friable and highly embryogenic, and such callus, termed herein"friable embryogenic callus", generally grows faster than the compactembryogenic callus. From either type of callus, phenotypically normalplants can be regenerated, and in both types of callus, somatic embryosare present in different stages of development. The appearance and finalmorphology of the two types of callus can differ in different monocotspecies, particularly in different cereal species. Nevertheless, the twotypes of callus can be readily distinguished from one another by personsskilled in the art of forming and manipulating tissue cultures ofdifferent monocot species.

In corn, compact embryogenic callus and friable embryogenic callus aremore familiarly known as type I callus and type II callus, respectively.Various distinguishing features in the structure and properties of typeI and type II corn calli are described in publications, such as:Armstrong and Phillips (1988) Crop Sci. 28:363; Springer et al (1979)Protoplasma 101:269; Fransz (1988) "Cytodifferentiation during callusinitiation and somatic embryogenesis in Zea mays L.", Ph.D. Thesis,University of Wageningen, The Netherlands; Ozias-Akins et al (1982)Protoplasma 110:95; Novak et al (1983) Maydica 28:381; Ho et al (1983)Protoplasma 118:169; Green et al (1975) Crop Sci. 15:417; Freeling et al(1976) Maydica 21:97; Lu et al (1982) Theor. Appl. Genet. 62:109; Vasilet al (1985) Protoplasma 127:1; Dunstan et al (1978) Protoplasma 97:251;Vasil et al (1982) Bot. Gaz. 143:454; Green (1983) In: Basic biology ofnew developments in biotechnology, Hollaender et al (eds) Plenum Press,New York, pp. 195-209; Vasil et al (1984) Am. J. Bot. 71:158; and Kamoet al (1985) Bot. Gaz. 146:327.

Type I corn callus is essentially white, pale white or yellowish andcompact in appearance, often has a nodular surface, and represents thegeneration and propagation of an organized set of tissues which isreflected in its nodular appearance. it is characterized by a highdegree of cellular association and differentiation and by variousstructures, such as roots, leafy structures and vascular elements.Somatic embryos can generally be recognized. The origin of regeneratedshoots is not always obvious and can apparently occur by both somaticembryogenesis and organogenesis. During somatic embryo development,embryoids can fuse and give rise to hard, white callus or can developinto secondary somatic embryos.

Type II corn callus is essentially soft, friable, white or pale-yellow,somewhat transparent in appearance and highly embryogenic. It growsrapidly and contains no vascular elements. Type II callus differs fromnon-embryogenic friable callus in containing numerous smooth andglobular embryoids that may possess a suspensor-like structure by whichthe embryoids are attached to the callus. The embryoids can develop intowell-organized somatic embryos.

Approximately the same distinguishing features, that are found in thetwo types of corn calli, can be used to distinguish between the compactembryogenic callus and the friable embyogenic callus of other monocotspecies, particularly cereal species such as rice (Kyozuka et al (1988)Theor. Appl. Genet. 76:887), wheat (Redway et al (1990) Theor. Appl.Genet. 76:609; Redway et al (1990) Plant Cell Reports 8:714), andbarley.

From monocotyledonous plants generally, the compact embryogenic callusof this invention can be obtained by in vitro culture of explant sourcessuch as immature zygotic embryos, mature seeds, leaf bases, anthers,microspores, young inflorescences, etc. In corn, the type I callus ismost efficiently generated from immature zygotic embryos. The compactembryogenic callus can be induced from the appropriate explants andmaintained in culture according to well-established methods (see Hodgeset al (1986) Bio/Technology 4:219). During maintenance of the callusculture, care has to be taken to select and subculture only theembryogenic sectors of the calli in which are the embryogenic cells.Such cells can generally be characterized as small, tightly packed,thin-walled, richly cytoplasmic, highly basophilic cells containing manysmall vacuoles, lipid droplets and starch grains (Vasil (1988) supra).The most convenient way to remove, from a plant, tissues that are knownto be capable of forming the compact embryogenic callus is by means ofdissection.

The competent cells of this invention can be obtained directly from amonocotyledonous plant by cutting from the plant, in a conventionalmanner, intact tissue that is capable of forming compact embryogeniccallus. The cells of such wounded intact tissue can then be stablytransformed. However, it is preferred that such wounded intact tissue becut into smaller fragments to wound further such tissue and provide morecompetent cells for transformation. The average maximum dimension of thetissue fragments is preferably 0.1 to 5 mm long, particularly 1 to 2.5mm long, more particularly 1.25 to 1.75 mm long. In this respect, thewounded intact tissue of this invention can be any piece of tissue thatis cut from the plant or any fragments thereof (e.g., cut pieces). Thus,the term "intact tissue" should be understood as referring to aggregatesof monocot plant cells that are obtained from a naturally occurringplant part, without a tissue-culturing stage in between.

It is believed that the mechanical disruption or wounding of the intacttissue and its individual cells, by cutting the intact tissue from theplant and possibly further cutting it so as to disrupt or wound itfurther, is generally sufficient to generate the competent cells of thisinvention. In this regard, the terms "mechanical disruption" and"wounding" are intended to encompass the significant damaging of thecell wall of one or more cells of the intact tissue in order to exposethe cell(s) and render the cell(s) open to insertion of a DNA fragmentin accordance with this invention. Thus, "mechanical disruption" or"wounding" in accordance with this invention is not limited to cuttingthe cell wall but includes other methods of physically removing one ormore portions of the cell wall or rendering the cell wall discontinuousin one or more places, such as by abrading, squeezing or striking thecell wall.

However, the mechanical disruption or wounding of the intact tissue inaccordance with this invention can be supplemented or even replaced by atreatment of the intact tissue with an enzyme or enzyme mixture todegrade the plant cell walls, especially when the intact tissue isrelatively large. The enzyme treatment can be carried out in aconventional manner. Preferably, the enzyme is applied to the intacttissue primarily to generate pores in its cell walls. It is thereforepreferred that the enzyme treatment be relatively short (e.g., from 1 to10 minutes depending upon the nature and the consistency of the intacttissue) so as not to cause a complete disruption of the tissue.Depending upon the type of plant, various enzymes or enzyme solutionscan be used such as those listed by Powell and Chapman (1985) "PlantCell Culture, A Practical Approach", R. A. Dixon ed., Chapter 3.

When the intact tissue, obtainable from the plant, is too small to bewounded (e.g., cut) or wounded intact tissue is too small to be furtherwounded (e.g., cut into smaller pieces), the enzyme treatment can beused to generate additional competent calls. Such an enzyme treatmentcan also be particularly useful, by itself, for forming competent cellsof this invention in embryos, particularly in immature zygotic embryosisolated from developing seeds and in mature zygotic embryos isolatedfrom mature (e.g., dry) seeds of, for example, corn. Embryos aregenerally not cut to remove them from seeds and generally cannot be cutinto significantly smaller fragments without destroying their ability togenerate compact embryogenic callus. Immature embryos are particularlyimportant in corn as they are the only convenient and reliable source ofcompact embryogenic callus. In rice and other monocots, mature embryoscan also be used. In this regard, for plants such as corn, it ispreferred that the intact tissue (e.g., immature corn embryos) have amaximum length of about 0.5 to 2 mm, preferably 0.5 to 1.5 mm, eventhough smaller lengths of 0.5 to 1 mm can be used.

In accordance with this invention, the intact tissue is also preferablysubjected to a period of, for example, about 15 minutes or more,preferably about 30 minutes to about 5 hours, particularly 2 to 3 hours,of preplasmolysis which involves placing the tissue in a conventionalhypertonic solution, such as the electroporation buffer discussed below.The purpose of this preplasmolysis treatment is to separate at leastpartly, in the cells of the intact tissue, their protoplasts, preferablyall or at least part of their cell membranes, from their cell walls.Such preplasmolysis is preferably carried out after any wounding of theintact tissue but before any enzyme treatment of the intact tissue. Whenthe intact tissue has already been degraded by an enzyme treatment, itis preferred that any subsequent preplasmolysis be only for a briefperiod, and after the enzyme treatment of immature embryos of corn, asdiscussed above, it is preferred that such preplasmolysis not be carriedout at all.

The competent cells of this invention can also be obtained by: culturingin vitro the intact tissue of this invention to produce compactembryogenic callus; and then cutting the callus into smaller fragments.The resulting callus fragments should comprise, wholly or at least inpart, the embryogenic sectors or parts of the callus. The callusfragments also preferably have an average maximum length of 0.5 to 2.5mm, particularly 1 to 2 mm, more particularly 1.25 to 1.75 mm, andpreferably have a minimum length of about 0.1 mm. To obtain sufficientamounts of compact embryogenic callus, it is preferred to propagate theprimary callus, as obtained from tissue explants, for at least one monthand to subculture the embryogenic sectors of such primary callus atleast once during this period. It is believed that the mechanicaldisruption or wounding of the embryogenic sectors of the compactembryogenic callus and their cells by, for example, cutting them isgenerally sufficient to generate the competent cells of this invention.However, the mechanical disruption of the callus may be supplemented orreplaced by an enzyme treatment to degrade the callus cell walls,especially when the compact embryogenic callus fragments remainrelatively large. This enzyme treatment can be carried out in aconventional manner. The enzyme treatment preferably serves primarily togenerate pores in the cell walls of the cells of the callus fragments,and it is therefore recommended that the enzyme treatment be relativelyshort, preferably from 1 to 10 minutes depending upon the consistency ofthe callus fragments, so as not to cause a complete disruption of thetissues. Depending upon the monocot plant, various enzymes or enzymesolutions can be used such as those listed by Powell and Chapman (1985)supra. Preferably, the compact embryogenic callus fragments are alsosubjected to a period (e.g., 2 to 3 hours) of preplasmolysis, asdiscussed above.

The wounded and/or degraded, intact tissue or compact embryogenic callusfragments, particularly their embryogenic sectors, obtained as describedabove, are then brought into contact with one or more DNA fragmentscontaining gene(s) of interest in order to transform their competentmonocot plant cells of this invention. It is preferred that at least oneof the genes of interest be adapted to serve as a selectable marker inthe resulting transformed monocot plant cells. It is believed thatdirect gene transfer, particularly electroporation, provides optimaltransformation efficiency. However, other known DNA transfer techniquescan be used such as direct gene transfer using polyethyleneglycol,bombardment with DNA-coated microprojectiles (i.e., biolistictransformation using, for example, a particle gun), andAgrobacterium-mediated transformation.

The compact embryogenic callus, used in carrying out the planttransformation method of this invention, can have certaincharacteristics of a friable embryogenic callus. In this regard, acompact embryogenic callus or a friable embryogenic callus can change orbe caused to change into a type of callus that has some of thecharacteristics of compact embryogenic callus as well as somecharacteristics of friable embryogenic callus. As a result, such anintermediate type of callus and embryogenic portions thereof cansometimes be transformed in accordance with this invention. Indeed,somatic embryos that develop on such an intermediate type of callus, aswell as on friable embryogenic callus, can be isolated and can bewounded and/or degraded and then transformed as described above. Thus,in carrying out the method of this invention, such somatic embryosobtained from an intermediate type callus or a friable embryogeniccallus can be regarded as equivalent to immature or mature zygoticembryos obtained from developing or mature seeds, particularly whenelectroporation is used as the means for transforming cells of thesomatic embryos.

In accordance with this invention, electroporation can be carried out ina conventional manner. In this regard, the wounded and/or degradedintact tissue or callus fragments, particularly meristematic orembryogenic sections thereof, quite particularly embryogenic sectionsthereof, can be transferred to a cuvette suitable for use in anelectroporation apparatus (e.g., as described by Dekeyser et al (1990)The Plant Cell 2:591). Preferably, about 10 to 500 mg, particularlyabout 50 to 200 mg, most particularly about 100 to 150 mg, of intacttissue or callus fragments per 200 μl of electroporation buffer aretransferred to the cuvette. For cereals, such as corn, (where it ispreferred to use intact enzyme-treated immature embryos), it ispreferred that about 10 to 500 embryos, particularly about 50 to 150embryos, more particularly about 75 to 125 embryos, in 200 μl ofelectroporation buffer are transferred to the cuvette. The DNA is thenadded to the cuvette, and the electroporation is carried out.Preferably, the DNA is coincubated (e.g., for about 1 hour) with theintact tissue or callus fragments prior to electroporation. It isbelieved that best results can be obtained with linear, rather thancircular, DNA of relatively small size, preferably smaller than about 20kb, especially smaller than 15 kb, particularly smaller than 10 kb,quite particularly smaller than 6 kb (e.g., down to about 2-3 kb). Inthis regard, multiple linear DNA fragments of different composition canbe used to transform the competent cells of this invention with multiplegenes of interests. Preferably, about 5 to 30 μg, particularly about10-25 μg, quite particularly about 20 μg, of DNA is added to the cuvettecontaining the intact tissue or callus fragments. Particularelectroporation conditions are not believed to be critical, and goodresults can be obtained, for example, with a discharge of one pulse witha field strength of 375 V/cm from a 900 μF capacitor using anelectroporation buffer containing 150 mM NaCl or 80 mM KCl (Dekeyser etal (1990) supra).

When the transformation (e.g., by electroporation) is completed, theintact tissue or callus fragments, containing the transformed monocotcells, are transferred to a suitable culture medium, preferably aselective medium when the transformed cells contain a selectable marker.This transfer should be as soon as possible after, preferablyimmediately after, the transformation event and especially within one tothree days after the transformation event. Preferably, the intact tissueor callus fragments transformed with a selectable marker are culturedusing conventional culture conditions and culture media (see, e.g.,references in Vasil (1988) supra) supplemented with a selective agent.The selection of the selective agent will depend on the selectablemarker used in the DNA fragments to transform the cells of the intacttissue or callus fragments, as discussed below. The concentration of theselective agent should provide a very high selective pressure on thetransformed cells so that only stable transformants, in which the DNAfragments containing the selectable marker are integrated, preferablyfully integrated, in the genome of the cells, survive and can beisolated. Although such transformed intact tissue or callus fragmentscan be cultured for a few days on non-selective medium, it is preferredthat they be transferred to selective medium as soon as possible andmaintained for a prolonged period (e.g., as long as six months),preferably at least one month, especially two to three months, toproduce significant amounts of transformed morphogenic callus, such astransformed compact embryogenic callus, which can be used to regeneratea phenotypically normal plant. It is also preferred that thehypertonicity of the medium be maintained for a limited time (e.g., upto two to three weeks), for instance by supplementing the medium withmannitol.

In accordance with this invention, any DNA fragment can be integrated inthe genome, particularly the nuclear genome, of a monocotyledonousplant. Generally, the DNA fragment contains a foreign or endogenous geneor other DNA sequence which is functional in the transformed plant cellsand confers an additional property to such cells and to plantsregenerated from the cells. To this end, the DNA fragment preferablycomprises one or more chimaeric genes which contain the followingoperably linked DNA sequences: 1) a promoter sequence capable ofdirecting expression of a coding sequence in the plant cell (a"promoter"); 2) a sequence (a "coding sequence") coding for a proteinwith a specific activity within the plant cell (a "protein ofinterest"): and 3) suitable 3' transcription regulation signals. Inorder to obtain the required functionality of the protein, it may alsobe necessary that the protein be targeted to one or more particularcompartments of the plant cell, such as the cytosol, mitochondria,chloroplasts or endoplasmatic reticulum. For targeting to the cytosol,the chimaeric gene, as described above, can be used as such. However fortargeting to the other compartments, it is required that there be anadditional sequence (a "targeting sequence") between the DNAfragments 1) and 2) of the chimaeric gene. If required, the chimaericgene can also contain transcriptional and/or translational enhancers,and the codon usage of the DNA sequences can be optimized for expressionin plant cells.

Chimaeric genes in accordance with this invention can be constructedaccording to well-established principles and techniques. In this regard,the various DNA sequences should be linked so that translation isinitiated at the initiation codon of the coding sequence of the protein(or of the targeting sequence when it is present).

It is believed that the various constitutive and organ- andtissue-specific promoters that are presently used to direct expressionof genes in transformed dicotyledonous plants will also be suitable foruse in transformed monocots of this invention. In this regard,particular plant cells can be transformed with a chimaeric genecomprising: a coding sequence encoding a protein of interest; andupstream (i.e., 5') thereof, either a foreign or an endogenous promotersuitable for expression of the coding sequence. Suitable foreignconstitutive promoters include: the promoter of the Cauliflower MosaicVirus ("CaMV") isolates CM1841 (Gardner et al (1981) Nucl. Acids. Res.9:2871) and CabbB-S (Franck et al (1980) Cell, 21:285) (the "35Spromoter") which directs constitutive expression of heterologous genes(Odell et al (1983) Nature 313:810); a related promoter (the "35S3promoter") which can be isolated from the CaMV isolate CabbB-JI (Hulland Howell (1978) Virology 86:482) and which differs from the 35Spromoter in its sequence (the sequence of the 35S3 promoter is disclosedin European patent publication ("EP") 359617) and in its greateractivity in transgenic plants (Harpster et al (1988) Mol. Gen. Genet.212:182); and the TR1' and the TR2' promoters which drive the expressionof the 1' and 2' genes, respectively, of the T-DNA of Agrobacterium(Velten et al (1984) EMBO J. 3:2723) and are wound-induced promoters.Suitable organ-specific, tissue-specific and/or inducible foreignpromoters are also known (see, e.g., references cited in Kuhlemeier etal (1987) Ann. Rev. Plant Physiol. 38:221) such as the promoters of thesmall subunit genes (such as the 1A gene) of 1,5-ribulose bisphosphatecarboxylase of Arabidopsis thaliana (the "ssu" promoter) which are lightinducible promoters (Krebbers et al (1988) Plant Mol. Biol. 11:745)active only in photosynthetic tissue; the anther-specific promotersdisclosed in EP 344029; and the seed-specific promoters of, for example,Arabidopsis thaliana (Krebbers et al (1988) Plant Physiol. 87:859).Promoters of particular usefulness for transforming monocots to renderthem male-sterile, as described in EP 344029, are the tapetum-specificpromoters PTA29, PTA26 and PTA13, particularly PTA29, of EP 344029.

Likewise, it is believed that known 3' transcription regulationsequences and polyadenylation signals used in transformed dicotyledonousplants can be used in transformed monocots of this invention. Such 3'transcription regulation signals can be provided downstream (i.e., 3')of the coding sequence. In this regard, a particular plant cell can betransformed with a chimaeric gene containing either foreign orendogenous transcription termination and polyadenylation signalssuitable for obtaining expression of the chimaeric gene. For example,the foreign 3' untranslated ends of genes, such as gene 7 (Velten andSchell (1985) Nucl. Acids Res. 13:6998), the octopine synthase gene(Gielen et al (1983) EMBO J. 3:835) and the nopaline synthase gene ofthe T-DNA region of Agrobacterium tumefaciens Ti-plasmid can be used.

For construction of a chimaeric gene which can be expressed in atransformed plant cell, preferably in its cytoplasm followed bytranslocation of its protein of interest to the cell's mitochondria,chloroplasts and/or lumen of the endoplasmatic reticulum, suitabletargeting sequences are known. Selection of such targeting sequences isnot believed to be critical, and a particular plant cell can betransformed with a chimaeric gene containing either a foreign orendogenous targeting sequence encoding a targeting peptide which willprovide translocation of the expression product of the gene. By"targeting peptide" is meant a polypeptide fragment which is normallyassociated, in an eucaryotic cell, with a chloroplast or mitochondrialprotein or subunit of the protein or with a protein translocated to theendoplasmatic reticulum and which is produced in a cell as part ofprecursor protein encoded by the nuclear DNA of the cell. The targetingpeptide is responsible for the translocation process of thenuclear-encoded chloroplast or mitochondrial protein or subunit into thechloroplast or the mitochondria or the lumen of the endoplasmaticreticulum. During the translocation process, the targeting peptide isseparated or proteolytically removed from the protein or subunit. Atargeting sequence can be provided in the chimaeric gene to express atargeting peptide which can translocate an expressed protein of interestwithin a transformed plant cell as generally described in Europeanpatent applications ("EPA") 85402596.2 and 88402222.9. A suitabletargeting peptide for transport into chloroplasts is the transit peptideof the small subunit of the enzyme 1,5-ribulose bisphosphate carboxylase(Krebbers et al (1988) Plant Mol. Biol. 11:745; EPA 85402596.2), butother chloroplast transit peptides, such as those listed by Watson(1984) Nucl. Acids Res. 12:5145 and Von Heijne et al (1991) Plant Mol.Biol. Rep. 9:104, can also be used. Suitable mitochondrial targetingpeptides are the mitochondrial transit peptides as described by Schatz(1987) Eur. J. Biochem. 165:1 and listed by Watson (1984) supra.Suitable targeting peptides that can translocate a protein of interestto the lumen of the endoplasmatic reticulum of a plant cell are, forinstance, the signal peptides described by Von Heijne (1988) Biochem.Biophys. Acta 947:307 and listed by Watson (1984) supra.

Coding sequences that can be used for the production of transgenicdicotyledonous plants are well known (see, for example, the codingsequences listed in Weising et al (1988) Annual Rev. Genet. 22:421), andit is believed that such coding sequences can be put to equally good usein transformed monocotyledonous plants in accordance with thisinvention. In this respect, the coding sequences can be either foreignor endogenous to the plants and can, for example, code for proteinswhich: are toxic to insect species, thus protecting the plants againstinsect attack (EP 193259, EP 305275 and EP 358557); protect the plantsagainst stress conditions (EP 359617); confer on the plants a resistanceor tolerance to specific herbicides (EP 242236); kill or disable plantcells in which the proteins are expressed so that, when the codingsequences are under the control of a male or female organ-specificpromoter, the proteins can render the plants respectively male sterile(EP 344029) or female sterile (EP 412006); can be extracted from theplants or selected plant organs and optionally be further processed sothat the plants can be used as sources of economically importantpeptides or proteins (EP 319353); or are enriched in nutritionallyimportant amino acids so that transformed plants or their organs, inwhich the proteins are expressed, can be used as food with enhancednutritional value for animals or humans (EP 318341).

Coding sequences of particular usefulness for transforming monocots torender them insect-resistant are the genes isolated from Bacillusthuringiensis ("Bt") strains and truncated portions thereof that codefor insecticidal crystal proteins and their insecticidal polypeptidetoxins (for a review, see: Hofte and Whiteley (1989) Microbial. Rev.53:242). The following Bt genes are believed to be particularlyimportant for insect control in cereals (e.g., corn, rice, wheat andbarley): the CryIAb gene (EP 193259) and CryIAc gene for control ofHelicoverpa species (e.g., H. zea and H. armigera); the CryIAb gene andthe CryIb gene (EP 358557) for control of Ostrinia species (e.g., O.nubilalis) in corn; the CryIAc gene for the control of Agrotis speciesin corn and wheat; and the CryID and CryIE genes (EP 358557) for thecontrol of Spodoptera species (e.g., S. frugiperda) in corn. To achievesufficient expression of such genes in tissues of transgenic plants, itis preferred that the genes be modified as described in PCT applicationPCT/EP 91/00733 (PCT publication WO 91/16432).

Selectable markers in accordance with this invention are chimaeric genesin which the coding sequences encode proteins which confer on the plantcells, in which they are expressed, resistance to a selectable agentsuch as an antibiotic and/or herbicide. Screenable markers in accordancewith this invention are chimaeric genes in which the coding sequencesencode proteins which confer of the plant cells, in which they areexpressed, a different appearance, such as a different color, makingplants transformed with the screenable marker separable manually. Theselection of a selectable or screenable marker, preferably a selectablemarker, for transforming a monocotyledonous plant in accordance withthis invention is not believed to be critical, and it is believed thatconventional selectable and screenable markers can be used (see, forexample, the markers listed in Weising et al (1988) supra). Examples ofsuitable coding sequences for selectable markers are: the neo gene (Becket al (1982) Gene 19:327) that codes for the enzyme neomycinphosphotransferase which confers resistance to the antibiotic kanamycin;the hyg gene (Gritz and Davies (1983) Gene 25:179) that codes for theenzyme hygromycine phosphotransferase which confers resistance to theantibiotic hygromycin; and the bar gene (EP 242236) that codes forphosphinothricin acetyl transferase which confers resistance to theherbicide phosphinothricin. In using a selectable marker gene coding fora protein that confers tolerance or resistance to a herbicide or otherselective agent that acts on chloroplast metabolism, such as the bargene, it is preferred that the marker gene be part of a chimaeric genetogether with a chloroplast targeting sequence as described above.Examples of suitable coding sequences for screenable markers are the gusgene (Jefferson et al (1986) Proc. Natl. Acad. Sci. USA 6:3901) encodingbeta-glucuronidase and the luciferase gene (Ow et al (1986) Science234:856).

As discussed above, the selection pressure, provided by the presence ofa selectable agent, should preferably be rather high during culturing oftransformed plant cells of this invention containing selectable markers.For example, when the neo gene is used as a selectable marker, kanamycinshould be used in concentrations of at least about 100-200 mg per liter,preferably at least about 200 mg per liter, in the culture medium. Suchhigh selection pressure should also be maintained for a prolonged time,for example, two to four months. It is believed, however, thatparticular selection pressures and durations are not critical and thatthe choice of selection pressures and their durations can be made in aconventional manner. However when the bar gene is used as a selectablemarker gene, phosphinothricin (PPT) is preferably used in concentrationsof 0.5 to 50, particularly 2 to 20, mg per liter of the culture medium.

Morphogenic sectors, preferably embryogenic sectors, of morphogeniccallus, preferably compact embryogenic callus, produced in a culture oftransformed cells of wounded and/or degraded intact tissue or woundedand/or degraded embryogenic sectors of compact embryogenic callus ofthis invention, can then be regenerated into phenotypically normal(e.g., mature and fertile) plants in a conventional manner (see, forexample, references in Vasil (1988) supra and Lazzeri and Lorz (1988)supra). The regenerated plants, thus obtained, will be transgenic andwill at least possess the selectable or screenable marker, preferablythe selectable marker, stably integrated into their nuclear genome. Thepresence and expression of other genes of interest can then be evaluatedin a conventional manner, such as by means of Southern blotting and/orby the polymerase chain reaction (Sambrook et al (1990) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory).

For the purposes of this invention, a phenotypically normal plant asproduced by the transformation and regeneration procedures of thisinvention should be understood as at least one plant that does notdiffer substantially from an untransformed plant of the same line in anyof its phenotypic characteristics except in those characteristics thatare added or changed due to the expression of the DNA fragmentsintroduced in the plant's genome during transformation. Of course, anyprocedure for transforming plants usually produces a number oftransgenic plants that display a variety of phenotypes, only some ofwhich are phenotypically normal as defined above.

The method of this invention can be applied to all monocotyledonousplant species from which compact morphogenic callus, such as compactembryogenic callus, can be obtained during in vitro culture of explantsderived from various explant sources such as immature and mature zygoticembryos, leaf bases, young inflorescences, etc. The method will beespecially useful for the transformation of economically importantgramineous crops, particularly the major cereals, such as corn, wheat,rice, oats, barley, sorghum, rye and millet. The resulting transgenicplants of this invention can be used to create, in a rapid and efficientmanner, novel lines and/or cultivars of high agronomic value. In thisregard, transgenic plants can be created in accordance with thisinvention that can be used as pollinator plants, for example, asfemale-sterile pollinator plants for the production of hybrid seed asdisclosed in EP 412006 (which is incorporated herein by reference).

This invention provides a rapid, efficient and reproducible method fortransforming monocotyledonous plants, using intact tissue or compactembryogenic callus to produce cultures of transformed morphogeniccallus, preferably compact embryogenic callus. This is surprising asneither intact tissue nor compact embryogenic callus has generally beenregarded as a suitable starting material for obtaining stabletransformants (see Vasil (1990) Bio/Technology 8:797). The use of intacttissue or compact embryogenic callus in accordance with this inventionis a distinct improvement on existing monocot transformation methods 7which have required the use of friable embryogenic callus, embryogeniccell suspension cultures and/or protoplasts that are competent for: 1)DNA uptake, 2) integrative transformation and 3) efficient andreproducible, monocotyledonous plant regeneration. Such requirements ofcompetence have, up to now, limited stable transformations of monocotsto plant lines with very specific tissue culture properties. In corn forexample, only certain lines, such as the inbred line A188, have had thecapacity to form enough type II callus (i.e., to form type II callus atfrequencies higher than 10%, up to, for example, 80% or more), fromwhich competent suspension cultures and/or protoplasts could be obtainedat appreciable frequencies. However, all such corn lines have been oflow agronomic value, so that transformations of economically valuablecorn lines have only been possible by laborious breeding programs inwhich appropriate tissue culture properties have been transferred to thevaluable corn lines from the transformable low value lines.

Because the method of this invention requires only a relatively shortperiod of in vitro culture, the method is far less time and laborconsuming than previous methods. The short tissue culture period alsoensures that the occurrence of somaclonal variation is reduced.

The method of this invention provides novel, phenotypically normal(e.g., fertile), transgenic monocotyledonous plants, particularlygramineous plants, quite particularly cereals, most particularly cornand rice, which are transformed with at least one (e.g., foreign) geneof interest, stably integrated into their nuclear genome. The method isbelieved to be independent of the genotype of the plant, beingtransformed, and capable of transforming cells of any plant, from whichcompact embryogenic callus can be obtained from at least one of itstissues. This makes it possible to transform the majority of monocotspecies and a substantial number of lines within each species. Moreover,the capacity to form compact embryogenic tissue can be transferred, bymeans of classical breeding programs, from one line that posesses suchcapacity to another line that does not.

The novel transgenic monocot plants of this invention regenerated fromtransformed morphogenic callus, particularly transformed compactembryogenic callus, are characterized by the fact that from suchmonocots, using conventional culture conditions as described, forexample, in Datta et al (1990) supra, Shimamoto et al (1989) supra,Hayashimoto et al (1990) supra, Gordon-Kamm et al (1990) supra, andFromm et al (1990) supra, it is practically impossible to obtainembryogenic suspension cultures and/or protoplasts or it is practicallyimpossible to obtain embryogenic suspension cultures and/or protoplastswhich have sufficient capability of being stably transformed and thenregenerated as phenotypically normal (e.g., fertile), transgenic plants.In regard to this second type of impossibility, it is not believedpractical to obtain embryogenic suspension cultures or protoplasts ofsuch monocots that: 1) have a high probability of being regenerable intophenotypically normal plants; 2) have a high probability of beingcompetent with respect to DNA uptake and integrative transformation ofthe so taken-up DNA; and 3) when so transformed, have a high probabilityof being regenerable into phenotypically normal, transgenic plants.

In particular, this invention provides novel transgenic rice plants ofrice lines, from which embryogenic suspension cultures (when obtainable)can generally be obtained, for example, according to the proceduresdescribed by Li et al (1990) Plant Mol. Biol. Report. 8:276, Datta et al(1990) Plant Sci. 67:83, and Datta et al (1990) Plant Cell Rep. 9:253,and protoplasts (when obtainable) can generally be obtained from theembryogenic suspension cultures, for example, according to theprocedures described by Li and Murai (1990) Plant Cell Rep. 9:216.However under conventional culture conditions as described, for example,in Shimamoto et al (1989) supra, Datta et al (1990) supra andHayashimoto et al (1990) supra, it is practically impossible toregenerate phenotypically normal (e.g., fertile) plants from embryogenicsuspension cultures or protoplasts of such rice lines.

This invention also provides novel transgenic corn plants of corn lines,from which embryogenic suspension cultures (when obtainable) cangenerally be obtained, for example, according to the proceduresdescribed by Shillito et al (1989) Bio/Technology 7:581, Prioli andSondahl (1989) Bio/Technology 7:589, Gordon-Kamm et al (1990) supra, andFromm et al (1990) supra, and protoplasts (when obtainable) cangenerally be obtained from such embryogenic suspension cultures, forexample, according to the procedures described by Shillito et al (1989)supra and Prioli and Sondahl (1989) supra. However under conventionalculture conditions as described, for example, by Shillito et al (1989)supra, Prioli and Sondahl (1989) supra, Gordon-Kamm et al (1990) supraand Fromm et al (1990) supra, it is practically impossible to regeneratephenotypically normal (e.g., fertile) plants from embryogenic suspensioncultures or protoplasts of such corn lines. Furthermore, such corn lineshave the capacity to form type I callus at high frequencies but do notpossess the ability to form type II callus at frequences higher than10%, particularly at frequencies higher than 1%, quite particularly atfrequencies higher than 0.1%, more quite particularly at frequencieshigher than 0.01. Type II corn callus is the only type of corn callustissue, from which embryogenic suspension cultures and regenerableprotoplasts can be suitably obtained that can be stably transformed, andthus, the inability to obtain type II callus for a particular corn linehas meant, up to now, that one could not regenerate phenotypicallynormal (e.g., mature), transgenic corn plants from transformed callus ofsuch corn line. The practical ability to obtain type II callus from aparticular corn line can be assessed by the general procedures describedby Gordon-Kamm et al (1990) supra and Fromm et al (1990) supra and thereferences mentioned therein. In making this assessment: callus culturescan be initiated from, for example, 1000 immature embryos of a cornline; the cultures can be maintained by subculturing every 3 weeks, andonly those of the cultures that most resemble typical type II callus canbe subcultured; and after 6 months, it can be determined at whatfrequencies a uniform type II callus is obtained.

More generally, to determine whether it is practical to obtainregenerable protoplasts from a specific line of a monocot species, thefollowing well known procedures can be followed. In this regard, it isbelieved that regenerable protoplasts are most efficiently and reliablygenerated from embryogenic suspension cultures which, for any specificmonocot, can be produced and maintained by conventional means. Theextent and quality of an embryogenic suspension culture is generallydependent on its genotype, and it is generally only worthwhile to formprotoplasts of a plant line, for transformation, if its embryogenicsuspension culture is capable of plant regeneration. Embryogenicsuspension cultures can generally be characterized as consisting of welldispersed, small groups of richly cytoplasmic embryogenic cells, asbeing free of callus tissues or organized meristems, as having celldoubling times of 27-32 hours, and as being capable of forming somaticembryos and plants (Vasil (1988) Bio/Technology 6:397). It can bedetermined whether an embryogenic suspension culture of a particularline of a monocot species is suitable for plant regeneration by platinga large number (i.e., at least 100) of cell aggregates on a suitableregeneration medium and determining what proportion of the aggregatesgive rise to phenotypically normal, fertile plants. If normal fertileplants are obtained from 50% or more of the cell aggregates, it isgenerally considered worthwhile to proceed with protoplast generation.However, a specific monocot line can be considered as not being suitablefor providing regenerable protoplasts suitable for plant transformationif, using conventional protoplast isolation, culture, and plantregeneration techniques: for every 10,000 protoplasts, no more thanabout 500, especially no more than about 100, particularly no more thanabout 10, quite particularly no more than about 1, phenotypically normal(e.g., fertile) plant(s) can be regenerated.

The Examples, which follow, illustrate this invention. Unless otherwiseindicated, all experimental procedures for manipulating recombinant DNAwere carried out by the standardized procedures described in Sambrook etal (1990) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory. Oligonucleotides were designed according to the generalrules outlined by Kramer and Fritz (1968) Methods in Enzymology 154:350and synthesized by the phosphoramidite method of Beaucage and Caruthers(1981) Tetrahedron Letters 22:1859 on an Applied Biosystems 380A DNAsynthesizer (Applied Biosystems B.V., Maarssen, Netherlands). Thefollowing bacterial strains and plasmids, used in the Examples, areavailable from the Deutsche Sammlung fur Mikroorganismen undZellkulturen ("DSM"), Mascheroder Weg 1B, Braunschweig, Germany:

    ______________________________________                                        Bacterial strain                                                                         plasmid   DSM No    Date of Deposit                                ______________________________________                                        E. coli WK6                                                                              pMa5-8    DSM 4567  May 3, 1988                                    E. coli WK6                                                                              pMc5-8    DSM 4566  May 3, 1988                                    ______________________________________                                    

EXAMPLE 1

Transformation of Corn with a Selectable Marker Gene by Electroporationof DNA into Zygotic Immature Embryos

Zygotic immature embryos of about 0.5 to 1 mm were isolated fromdeveloping seeds of two corn inbred lines, Pa91 and H99. The freshlyisolated embryos were enzymatically treated for 1-2 minutes with anenzyme solution II (0.3% macerozyme (Kinki Yakult, Nishinomiya, Japan)in CPW salts (Powell & Chapman (1985) "Plant Cell Culture, A PracticalApproach", R. A. Dixon ed., Chapter 3) with 10% mannitol and 5 mM2-[N-Morpholino]ethane sulfonic acid (MES), pH 5.6). After 1-2 minutesincubation in this enzyme solution, the embryos were carefully washedwith N6aph solution (macro- and micro-elements of N6 medium (Chu et al(1975) Sci. Sin. Peking 18:659) supplemented with 6 mM asparagine, 12 mMproline, 1 mg/l thiamine-HCl, 0.5 mg/l nicotinic acid, 100 mg/l caseinhydrolysate, 100 mg/l inositol, 30 g/l sucrose and 54 g/l mannitol).After washing, the embryos were incubated in the maize electroporationbuffer, EPM-NaCl (150 mM NaCl, 5 mM CaCl₂, 10 mM HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 0.425 Mmannitol, pH 7.2). Approximately 100 embryos in 200 μl EPM-NaCl wereloaded in each cuvette. About 20 μg of a plasmid DNA, pDE108 linearizedwith HindIII, were added per cuvette. pDE108 is a 5399 bp plasmid, theentire sequence of which is set forth in Seq. Id. No. 1 and whichcontains a chimaeric gene comprising the kanamycin resistance gene (neo)under the control of the 35S3 promoter (EP 359617).

After 1 hour DNA incubation with the explants, the cuvettes weretransferred to an ice bath. After 10 minutes incubation on ice, theelectroporation was carried out: one pulse with a field strength of 375V/cm was discharged from a 900 μF capacitor. The electroporationapparatus was as described by Dekeyser et al (1990) The Plant Cell2:591. Immediately after electroporation, fresh liquid N6aph substratewas added to the explants in the cuvette, after which the explants wereincubated for a further 10 minute period on ice.

Afterwards, the embryos were transferred to Mah1 VII substrate (macro-and micro-elements and vitamins of N6 medium supplemented with 100 mg/lcasein hydrolysate, 6 mM proline, 0.5 g/l MES, 1 mg/l2,4-dichlorophenoxyacetic acid (2,4-D) and 2% sucrose solidified with0.75 g/l MgCl₂ and 1.6 g/l Phytagel (Sigma Chemical Company, St Louis,Mo. U.S.A.), pH 5.8) and supplemented with 0.2M mannitol. After 3 daysfor line H99 and 2 days for line Pa91, the embryos were transferred tothe same substrate supplemented with 200 mg/l kanamycin. Afterapproximately 14 days, the embryos were transferred to Mah1 VIIsubstrate without mannitol, supplemented with kanamycin. The embryoswere further subcultured on this selective substrate for approximately 2months with subculturing intervals of about 3 weeks. The inducedembryogenic tissue was carefully isolated and transferred to MS medium(Murashige and Skoog (1962) Physiol. Plant 15:473) supplemented with 5mg/l 6-benzylaminopurine for line H99 and 5 mg/l zeatin for line Pa91.The embryogenic tissue was maintained on this medium for approximately14 days and subsequently transferred to MS medium without hormones and6% sucrose for line H99 and 3% sucrose for line Pa91. Developing shootswere transferred to 1/2 MS medium with 1.5% sucrose for furtherdevelopment to normal plantlets. These plantlets were transferred tosoil and cultivated in the greenhouse.

EXAMPLE 2

Characterization of the Transformed Corn Plants of Example 1

Seventeen plants from Example 1 were analysed for the presence of thechimaeric neo gene by means of the polymerase chain reaction (PCR). DNAwas prepared according to the protocol described by Dellaporta et al(1983) Plant Mol. Biol. Reporter 1:19, adapted for application to tissueamounts of about 10 to 20 mg. For each plant, such an amount of tissuewas macerated in extraction buffer in a microfuge tube. A 706 bpfragment, corresponding to part of the coding sequence of the neo gene,was amplified with the polymerase chain reaction according to theprotocol described by Sambrook et al (1990) supra, using oligonucleotideprobes complementary to the sequences of plasmid pDE108 from nucleotide1384 to 1406 and 2089 to 2067 (numbering as in Seq. Id. No. 1). Intotal, 35 cycles with an annealing temperature of 50° C. were carriedout. The final DNA was analysed by electrophoresis on a 1.5% agarosegel. A 706 bp fragment could be identified in a total of 13 plants. Oneof the positive plants died at a later stage.

Activity of the expression product of the neo gene (i.e., neomycinphosphotransferase II (NPTII)) was assayed in 9 of the plants asfollows. Crude extracts were prepared by grinding plant tissue inextraction buffer (McDonnell et al (1987) Plant Molecular Biol. Reporter5:380). The extracts were then subjected to non-denaturingpolyacrylamide gel electrophoresis according to the procedure describedby Reiss et al (1984) Gene 30:211. NPTII activity was then assayed by insitu phosphorylation of kanamycin using [gamma-³² P]ATP as a substrate(McDonnell et al (1987) supra). NPTII activity was found in 8 of theplants that were examined (FIG. 1).

One of the plants (H99-M148-1), that was found to be positive on boththe PCR and NPTII assay, was further analyzed by means of Southernhybridization. Genomic DNA was prepared from plant tissue according tothe protocol described by Dellaporta et al (1983) supra supplemented bya treatment with RNase to remove remaining RNA. A non-transformed H99plant was used as a control. Samples of the DNA were digested with oneof the following restriction enzymes: BglII, EcoRI, EcoRV, HindIII,BamHI, PvuI, PvuII or PstI and subjected to horizontal agaroseelectrophoresis. Southern transfer to Hybond N+ (Amersham InternationalPLC, Amersham, United Kingdom) membranes by means of the "alkaliblotting of DNA" protocol and the subsequent hybridization wereperformed as recommended by the manufacturer (Amersham Hybond-N+leaflet). Radioactive probes were prepared with the multi-prime DNAlabelling kit (Amersham) according to the protocol supplied by themanufacturer which was derived from published procedures (Feinberg andVogelstein (1983) Anal. Biochem. 132:6). As a probe, a 1184 bpEcoRI-HindIII fragment derived from another plasmid was used. Thesequence of this plasmid is given in Seq. Id. No. 2. The bandingpatterns (e.g., see FIG. 2) showed that at least the chimaeric neo genewas integrated into the plant genomic DNA.

Further analysis of this transformed plant (H99-m148-1) showed that itcarries two almost intact copies of the plasmid pDE108 and part of athird rearranged copy. The two almost complete copies are apparentlyinserted in the plant genome in a head to tail concatamer configuration.However, some rearrangements must have occurred as an additional NcoIsite and an additional BglII site were created, while the HindIII site(used for linearization of pDE108 prior to electroporation) at thejunction of the two copies was lost. Sequencing of the junction of thetwo plasmid copies, as integrated in the plant genome, revealed thatonly the protruding 5' termini of the HindIII site are missing, thuscreating a NcoI site as follows:

    ______________________________________                                        AGCCA     + AGCTTGGCG = AGCCATGGCG                                            TCGGTTCGA       ACCGC   TCGGTACCGC                                            ______________________________________                                    

(the lost bases are underlined, and the created NcoI site at thejunction is highlighted). Additional analysis showed that no or very fewplasmid DNA sequences around the HindIII sites, flanking the plantgenome, were lost. Although the other plants were not tested in thisway, the PCR and NPTII assays showed that the chimaeric genes arepresent and expressed.

The mature transformed plants were fertile and phenotypically completelynormal. The plant that was previously assayed by Southern hybridizationwas used as pollinator plant in three crossings with untransformedplants (two from corn inbred line H99 and one from corn inbred linePa91). A total of 44 of the plants of the F1 progeny were assayed forNPTII activity as described above, and twenty of them were found to bepositive. This does not differ significantly from the 1:1 ratio expectedunder normal Mendelian segregation assuming that the transformedpollinator plant had one active copy (or alternatively, multiple closelylinked active copies) of the chimaeric neo gene (X² =0.36).

EXAMPLE 3

Transformation of Corn with a Selectable Marker Gene by Electroporationof DNA into Type I Callus Derived from Immature Zygotic Embryos

Immature zygotic embryos of about 0.5 to 1 mm in length were isolatedfrom developing seeds of the corn inbred line Pa91 and cultured on Mah1VII substrate with subsequent subculture intervals of about 14 days.Embryogenic tissue was carefully dissected out from developing type Icallus. The embryogenic tissue in EPM (EPM-NaCl without NaCl) was thenfinely cut in fragments with a maximum length of about 1.5 mm. Theresulting callus fragments were preplasmolysed for three hours in thisbuffer. After three hours, the callus fragments were transferred toEPM-NaCl. About 100-150 mg of callus fragments were transferred to 200μl EPM-NaCl per cuvette. 20 μg DNA of plasmid pDE108 (Seq. Id. No. 1),linearized with HindIII, was added to each cuvette. The DNA wasincubated with the callus fragments for one hour, after which thecuvettes were transferred to an ice bath.

After 10 minutes incubation on ice, the electroporation was carried out:one pulse with a field strength of 375 V/cm was discharged from a 900 μFcapacitor. The electroporation apparatus was as described by Dekeyser etal (1990) supra. Immediately after electroporation, fresh liquid N6aphsubstrate, supplemented with 6 mM asparagine, 12 mM proline, 1 mg/lthiamine-HCl, 0.5 mg/l nicotinic acid, 100 mg/l casein hydrolysate, 100mg/l inositol, 30 g/l sucrose and 54 g/l mannitol, was added to thecallus fragments which were then further incubated for 10 minutes onice.

After one day culture in liquid N6aph substrate supplemented with 1 mg/l2,4-D, the callus fragments were transferred to Mah1 VII substratesupplemented with 0.2M mannitol and 200 mg/l kanamycin. Fourteen dayslater, the callus fragments were subcultured on the same selectivesubstrate but without mannitol and further cultured on this substratefor about 2 months with subculturing intervals of about 3 weeks. Theembryogenic sectors of the resulting calli were isolated from the slimytissue and transferred to MS substrate (Murashige and Skoog (1962)Physiol. Plant 15:473) with 3% sucrose and supplemented with 5 mg/lzeatin to germinate. Tissue was maintained on this medium forapproximately 2 weeks and subsequently transferred to MS medium with 3%or 6% sucrose. Shoots that developed on this substrate were transferredto half-strength MS medium with 1.5% sucrose for further development tonormal plantlets. These plantlets were transferred to soil andcultivated in the greenhouse.

EXAMPLE 4

Characterization of the Transformed Corn Plants of Example 3

Twenty nine plants from Example 3 were analysed for the presence of thechimaeric neo gene by means of the polymerase chain reaction. DNA wasprepared according to Dellaporta et al (1983) Plant Mol. Biol. Reporter1:19, adapted for application to tissue amounts of about 10 to 20 mg.For each plant, such an amount of tissue was macerated in extractionbuffer in a microfuge tube. A 706 bp fragment, corresponding to part ofthe coding sequence of the neo gene, was amplified with the polymerasechain reaction according to the protocol described by Sambrook et al(1990) supra, using oligonucleotide probes complementary to thesequences of plasmid pDE108 from nucleotide 1384 to 1406 and 2089 to2067 (numbering as in Seq. Id. 1). In total, 35 cycles with an annealingtemperature of 50 C. were carried out. The final DNA was analysed byelectrophoresis on a 1.5% agarose gel. A 706 bp fragment could beidentified in a total of 14 plants. One of the positive plants died at alater stage.

Activity of the NPTII expression product of the neo gene was assayed in24 of the plants as follows. Crude extracts were prepared by grindingplant tissue in extraction buffer (McDonnell et al (1987) PlantMolecular Biol. Reporter 5:380). The extracts were then subjected tonon-denaturing polyacrylamide gel electrophoresis according to theprocedure described by Reiss et al (1984) Gene 30:211. NPTII activitywas then assayed by in situ phosphorylation of kanamycin using[gamma-32P]ATP as a substrate (McDonnell et al, supra). NPTII activitywas found in 14 of the plants that were examined (FIG. 3). Two plantsthat were NPTII positive scored negative in a PCR assay.

Two of the plants (Pa91-M146-2 and Pa91-M149-1), that were found to bepositive on both the PCR and NPTII assays, were further analyzed bymeans of Southern hybridization. Genomic DNA was prepared from planttissue according to Dellaporta et al (1983) supra, supplemented by atreatment with RNase to remove remaining RNA. A non-transformed Pa91plant was used as control. Samples of the DNA were digested with one ofthe following restriction enzymes: BglII, EcoRI, EcoRV, HindIII, BamHI,PvuI, PvuII or PstI and subjected to horizontal agarose electrophoresis.Southern transfer to Hybond N+ membranes by means of the "alkaliblotting of DNA" protocol and the subsequent hybridization wereperformed as recommended by the manufacturer (Amersham). Radioactiveprobes were prepared with the multi-prime DNA labelling kit (Amersham)according to the protocol supplied by the manufacturer which was derivedfrom published procedures (Feinberg and Vogelstein (1983) Anal. Biochem.132:6). As a probe, a 1184 bp EcoRI-HindIII fragment derived fromanother plasmid was used. The sequence of this plasmid is given in Seq.Id. No. 2. The banding patterns (e.g., see FIG. 4) showed that at leastthe chimaeric neo gene was integrated into the plant genomic DNA.

Further analysis of one of the transformed plants (Pa91-M146-2) showedthat it carried two almost intact copies of the plasmid pDE108 in a headto tail configuration. The HindIII site (used for linearization ofpDE108 prior to electroporation) at the junction of the two copies waslost. Sequencing of the junction of the two plasmid copies, asintegrated in the plant genome, revealed that the protruding 5' terminiof the HindIII site plus one base downstream of one of the HindIII sitesare missing as follows:

    ______________________________________                                        AGCCA     + AGCTTGGCG = AGCCAGGCG                                             TCGGTTCGA       ACCGC   TCGGTCCGC                                             ______________________________________                                    

(the lost bases are underlined). Additional analysis showed that no orvery few plasmid DNA sequences around the HindIII sites, flanking theplant genome, were lost. Although the other plants were not tested inthis way, the PCR and NPTII assays showed that the chimearic genes arepresent and expressed.

The adult plants were fertile and phenotypically completely normal. Oneof the plants, previously assayed by Southern hybridization, was used asa pollinator plant in a crossing with an untransformed plant from thecorn inbred line H99. A total of 20 plants of the F1 progeny wereassayed for NPTII activity as described above, and six of them werefound to be positive. This does not differ significantly from the 1:1ratio expected under normal Mendelian segregation assuming that thetransformed pollinator plant had one active copy of the chimaeric neogene (X² =3.2).

EXAMPLE 5

Transformation of Corn with a Male-sterility Gene and a SelectableMarker by Gene Electroporation of DNA into Zygotic Immature Embryos

Zygotic immature embryos of about 1 to 1.5 mm were isolated fromdeveloping seeds of corn inbred line H99. Freshly isolated embryos wereenzymatically treated and washed as described in Example 1. Afterwashing, the embryos were loaded in the maize electroporation buffer,EPM-KCl (80 mM KCl, 5 MM CaCl₂, 10 mM HEPES and 0.425 M mannitol, pH7.2). Approximately 100 embryos in 200 μl EPM-KCl were loaded in eachcuvette. About 20 μg of a plasmid DNA, pVE107 linearized with HindIII,were added per cuvette. pVE107 is a 6659 bp plasmid which is obtained byligation of the 1287 bp EcoRV-EcoRI fragment of pTTM8 (EP 344029; Seq.Id. No. 3) to the large XbaI (filled-in with Klenow)-EcoRI fragment ofplasmid pDE108 (Seq. Id. No. 1). pVE107 contains: a chimaeric genecomprising the kanamycin resistance gene (neo) under the control of the35S3 promoter; and another chimaeric gene comprising the barnase gene(Hartley (1988) J. Mol. Biol. 202:913) under the control of thetapetum-specific promoter of the TA29 gene of Nicotiana tabacum (EP344029).

All vector constructions involving fragments of the barnase gene werecarried out in E. coli strain WK6 containing the plasmid pMc5BS. pMc5BScontains the barstar gene (encoding an inhibitor of barnase) under thecontrol of the tac promoter (De Boer et al (1983) Proc. Natl. Acad. Sci.USA 80:21). This plasmid is constructed by: cloning the EcoRI-HindIIIfragment of plasmid pMT416 (see Hartley (1988) supra) into the EcoRI andHindIII sites of plasmid pMc5-8 (DSM 4566); and then deleting thesequence, starting with the initiation codon of the phoA signal sequenceand ending with the last nucleotide before the translation initiationcodon of the barstar coding region, by means of a looping-outmutagenesis procedure as generally described by Sollazo et al (1985)Gene 37:199.

After a 1 hour DNA incubation with the explants, the cuvettes weretransferred to an ice bath. After 10 minutes incubation on ice, theelectroporation was carried out as described in Example 1. Immediatelyafter electroporation, fresh liquid N6aph substrate was added to theexplants in the cuvette, after which the explants were incubated for afurther 10 minute period on ice.

Afterwards, the embryos were transferred to Mah1 VII substratesupplemented with 0.2 M mannitol and 200 mg/l kanamycin. Afterapproximately 14 days, the embryos were transferred to Mah1 VIIsubstrate without mannitol but with the same selective agent, kanamycin.The embryos were further subcultured on this selective substrate forapproximately 2 months, with subculturing intervals of about 3 to 4weeks. The induced embryogenic tissue was carefully isolated andtransferred to MS medium (Murashige and Skoog (1962) supra) supplementedwith 5 mg/l 6-benzylaminopurine. The embryogenic tissue was maintainedon this medium for approximately 14 days and subsequently transferred toMS medium without hormones and sucrose. Developing shoots weretransferred to 1/2 MS medium with 1.5% sucrose for further developmentto normal plantlets. These plantlets were transferred to soil andcultivated in the greenhouse.

EXAMPLE 6

Characterization of the Transformed Corn Plants of Example 5

Seven plants from Example 5, designated RZM19-2, RZM19-3, RZM19-4,RZM19-5, RZM19-6, RZM19-7 and RZM19-8, were derived from the sameembryogenic callus clump. They were subjected to extensive Southernanalysis. In this regard, BamHI-NcoI digested genomic DNA of the plantswas probed with pVE107 and with the small EcoRV-XbaI fragment of pTTM8(containing PTA29-barnase; see Seq. Id. No. 3). In all plants, thestrongest detected band was the expected 1400 bp fragment. However, thepattern found in these and other southern blots was very complex andindicated that transformation had resulted in many insertions of all orpart of pVE107 into the plants' genomes. Some of the inserted copies ofpVE107 were apparently incomplete and/or had undergone rearrangements.However, the same complex integration pattern was found in all sevenplants. This could be explained by the fact that the seven transformantswere all derived from one embryogenic callus clump.

The transformed plants were male sterile but otherwise phenotypicallycompletely normal; female fertility, for instance, was normal. Thespikelets of the male flowers were of about normal length but were verythin and appeared to be empty, and they never opened. A detailedanalysis showed that the anthers were reduced to almost microscopicstructures. This phenotype indicates not only that at least one copy ofthe barnase gene was expressed but also that it was selectivelyexpressed in some or all of the tissues of the anthers.

Transformant RZM19-3 was pollinated with pollen from an untransformedH99 plant, and 53 progeny plantlets were recovered. Of these 53plantlets, 32 (60%) scored positive in a NPTII assay, while 21 (40%)were NPTII negative. This proportion in the F1 progeny does not differsignificantly from the 1:1 ratio expected under normal Mendeliansegregation assuming that the transformed female parent had one activecopy of the chimaeric neo gene (X² =2.28). The NPTII negative progenywere male fertile, while the NPTII positive progeny were male sterile.

31 NPTII positive progeny plants were subjected to Southern analysis. 28of these plants displayed the same integration pattern as that of theoriginal transformant, RZM19-3, from which they were derived. 3 plantshad a slightly altered pattern.

EXAMPLE 7

Transformation of Corn with a Male-sterility Gene and a HerbicideResistance Gene by Electroporation of DNA into Zygotic Immature Embryos

Zygotic embryos of corn inbred line H99 were isolated, enzymaticallytreated, washed, and loaded in electroporation buffer as described inExample 5. Approximately 100 embryos in 200 μl EPM-KCl were loaded ineach cuvette. About 20 μg of a plasmid DNA, pVE108 linearized withHindIII, was added per cuvette. pVE108 is a 5620 bp plasmid which isobtained by ligation of the 1287 bp EcoRV-EcoRI fragment of pTTM8 (EP344029; Seq. Id. No. 3) to the large EcoRI-StuI fragment of plasmidpDE110 (Seq Id. No. 4). pVE108 contains: a chimaeric gene comprising thebar gene (EP 242236), encoding phosphinothricin acetyl transferase (PAT)and conferring resistance to an herbicidal glutamine synthetaseinhibitor such as phosphinothricin (PPT), under the control of the 35S3promoter; and another chimaeric gene comprising the barnase gene(Hartley (1988) supra) under the control of the tapetum-specificpromoter of the TA29 gene (EP 344029) of N. tabacum. All vectorconstructions involving DNA fragments comprising the barnase gene werecarried out in E. coli strain WK6 containing the plasmid pMc5BS ofExample 5.

After a 1 hour DNA incubation with the explants, the cuvettes weretransferred to an ice bath. After 10 minutes incubation on ice, theelectroporation was carried out as described in Example 1. Immediatelyafter electroporation, fresh liquid N6aph substrate was added to theexplants in the cuvette, after which the explants were incubated for afurther 10 minute period on ice.

Afterwards, the embryos from one electroporation experiment weretransferred to Mah1 VII substrate supplemented with 0.2 M mannitol and 2mg/l PPT. After approximately 14 days, the embryos were transferred toMh1 VII substrate (Mah1 VII substrate of Example 1 but without prolineand casein hydrolysate) supplemented with 2 mg/l PPT but withoutmannitol. After approximately 4 weeks, the embryos were subcultured foranother month on Mh1 VII substrate supplemented with 10 mg/l PPT. Theinduced embryogenic tissue was carefully isolated and transferred to MSmedium supplemented with 5 mg/l 6-benzylaminopurine. The embryogenictissue was maintained on this medium for approximately 14 days andsubsequently transferred to MS medium without hormones and sucrose.Developing shoots were transferred to 1/2 MS medium with 1.5% sucrosefor further development to normal plantlets. These plantlets survived anin vitro spraying with doses of BASTA^(R) (Hoechst AG, Frankfurt amMain, Germany) corresponding to 2 l/ha. These plantlets were thentransferred to soil and cultivated in the greenhouse, and two of thetransformed plantlets, designated RZM35-1 and RZM35-18, were furthercharacterized (see Example 8).

The embryos from a second eletroporation experiment were transferred toMh1 VII substrate supplemented with 2 mg/l PPT and 0.2 M mannitol. Afterabout 14 days, the embryos were transferred to Mh1 VII substratesupplemented with 2 mg/l PPT but without mannitol. After approximatelyanother three weeks, the embryos were transferred to Mh1 VII substratesupplemented with 10 mg/l PPT but without mannitol. After another threeweeks, the induced embryogenic tissue was carefully isolated andtransferred to MS medium supplemented with 2 mg/l PPT and 5 mg/l6-benzylaminopurine. The embryogenic tissue was maintained on thismedium for approximately 14 days and subsequently transferred to MSmedium without hormones, sucrose or PPT. Developing shoots weretransferred to 1/2 MS medium with l.5% sucrose for further developmentto normal plantlets. The resulting plantlets were transferred to soiland cultivated in the greenhouse, and three of the transformedplantlets, designated RZM34-1, RZM34-12, and RZM34-14, were furthercharacterized (see Example 8).

EXAMPLE 8

Characterization of the Transformed Corn Plants of Example 7

RZM34-1, RZM34-12, RZM34-14, RZM35-1, and RZM35-18 of Example 7 weregrown in the greenhouse. Activity of the expression product of the bargene in leaves of the plants was assayed as follows in a "PAT assay".100 mg of leaf tissue from each plant, together with 50 mg ofacid-treated sea sand (Merck, Darmstadt, Germany) and 5 mgpolyvinylpolypyrrolidone (PVPP), were ground in an Eppendorf tube with aglass rod in 50 μl of extraction buffer (25 mM Tris-HCL pH 7.5, 1 mM Na₂-EDTA (ethylenediaminetetraacetic acid disodium salt), 0.15 mg/mlphenylmethylsulfonylfluoride (PMSF), 0.3 mg/ml dithiothreitol (DTT), and0.3 mg/ml bovine serum albumin). The extract was centrifuged in amicrofuge for 5 minutes at 16000 rpm. The supernatant was recovered anddiluted ten times with TE 25/1 (25 mM Tris-HCL pH 7.5, 1 mM Na₂ -EDTA.To twelve μl of the diluted extract was then added: 1 μl of 1 mM PPT inTE 25/1, 1 μl of 2 mM ActeylCoenzyme A in TE 25/1, and 2 μl of [¹⁴C]AcetylCoenzym A (60 mCi/mmol, 0.02 mCi/ml, [NEN Research Products,DUPONT, Wilmington, Del., U.S.A.). The reaction mixture was incubatedfor 30 minutes at 37° C. and spotted on a aluminium sheet silicagel 60t.l.c. plate with concentrating zone (Merck). Ascending chromatographywas carried out in a 3 to 2 mixture of 1-propanol and NH₄ OH (25% NH₃).¹⁴ C was visualized by overnight autoradiography (XAR-5 Kodak film).

The tolerance to the herbicide BASTA® was tested by brushing a smallarea near the top of one leaf per plant with a 1% solution of theherbicide and observing the damage symptoms at and near the brushedsites. While RZM34-1, RZM35-1 and RZM35-18 showed no damage symptoms atall, RZM34-12 and RZM34-14 displayed slight browning and drying-out ofthe brushed site.

RZM34-1, RZM34-12, RZM34-14, RZM35-1 and RZM35-18 were also shown to bemale sterile. The phenotype of each of these plants was identical tothat described for the transformants of Example 5 which were analyzed inExample 6.

Southern analysis showed RZM35-1 and RZM35-18 to have an identicalintegration pattern, with only one copy of plasmid pVE108 being presentin the genome of each. A small part of the plasmid DNA sequence adjacentto the HindIII site (used for linearization prior to electroporation)seemed to be absent in the integrated copy. Southern analysis ofRZM34-1, RZM34-12 and RZM34-14 showed that each of these plants probablyhas two or three copies of part or all of pVE108 integrated into itsgenome. The copies are most likely not inserted in a concatemerconfiguration.

Transformants RZM35-1 and RZM34-1 were pollinated with pollen from anuntransformed H99 plant and progeny plantlets were recovered. From the35 plantlets recovered from RZM35-1, 16 (46%) scored positive in a PATassay, while 19 (54%) were PAT negative. This proportion in the F1progeny does not differ significantly from the 1:1 ratio expected undernormal Mendelian segregation of one active copy of the chimaeric bargene (X² =0.26).

From the 34 plantlets recovered from RZM34-1, 19 (56%) scored positivein a PAT assay, while 15 (44%) were PAT negative. This proportion in theF1 progeny does not differ significantly from the 1:1 ration expectedunder normal Mendelian segregation assuming that the transformed femaleparent had one active copy, or alternatively multiple active, butclosely linked copies, of the chimaeric bar gene (X² =0.47).

EXAMPLE 9

Transformation of Rice with a Herbicide Resistance Gene byElectroporation of DNA into Compact Embryogenic Callus Derived from DrySeeds

Dehusked mature seeds of the rice cultivar Nipponbare weresurfaced-sterilized, placed on solid 2N6 medium (N6 medium (Chu et al(1975) supra), supplemented with 0.5 mg/l nicotinic acid, 0.5 mg/lpyridoxine-HCl, 1.0 mg/l thiamine-HCl, 2.0 mg/l 2, 4-D, 30 g/l sucrose,and 2.0 g/l Phytagel, pH 5.8), and cultured at 27° C. in the dark.Callus developed from the scutella of the embryos within 3-4 weeks.Embryogenic portions of primary callus were transferred to N67 medium(N6 medium (Chu et al (1975) supra), supplemented with 0.5 mg/lnicotinic acid, 0.5 mg/l pyridoxine-HCl, 1.0 mg/l thiamine-HCl, 2.0 g/lcasamino acids (vitamin assay, Difco), 1.0 mg/l 2,4-D, 0.5 mg/l6-benzylaminopurine, 20 g/l sucrose, 30g/l sorbitol, and 2.0 g/lPhytagel, pH 5.8) for propagation into compact embryogenic callus.

Three to four weeks after subculture, the embryogenic callus was usedfor transformation experiments. The callus was cut into fragments with amaximum length of about 1.5 to 2 mm. The callus pieces were washed twicein EPM and then preplasmolyzed in this buffer for 30 minutes to 3 hoursat room temperature (25° C.). Then, the callus fragments were washedtwice with EPM-KCl and transferred to electroporation cuvettes. Eachcuvette was loaded with about 150 to 200 mg of callus fragments in 100to 200 μl EPM-KCl. 10 to 20 μg of a plasmid DNA, either circular pDE110or pDE110 linearized with HindIII or EcoRI, were added per cuvette.pDE110 is a 4883 bp plasmid, the entire sequence of which is set forthin Seq. Id. No. 4 and which contains a chimaeric gene comprising the bargene under the control of the 35S3 promoter.

The DNA was incubated with the callus fragments for about 1 hour at roomtemperature. Electroporation was then carried out as described inExample 1. After electroporation, liquid N67 medium without casaminoacids was added to the callus fragments. The callus fragments were thenplated on solid N67 medium without casamino acids but supplemented with5, 10 or 20 mg/l PPT and were cultured on this selective tedium at 27°C. under a light/dark regime of 16/8 hours for about 4 weeks. DevelopingPPT-resistant calli were isolated and subcultured for about two to threeweeks onto fresh N67 medium without casamino acids but containing 5 mg/lPPT. Thereafter, selected PPT-resistant calli were transferred to plantregeneration medium N6M25 (N6 medium (Chu et al (1975) supra),supplemented with 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine-HCl, 1.0mg/l thiamine-HCl, 288 mg/l aspartic acid, 174 mg/l arginine, 7.0 mg/lglycine, 1.0 mg/l )-naphthalenacetic acid (NAA), 5.0 mg/l kinetin, 20g/l sucrose and 2.0 g/l Phytagel, pH 5.8) supplemented with 5 mg/l PPT.Plantlets developed within approximately 1 month and were thentransferred to hormone-free N6 medium (Chu et al (1975) supra),supplemented with 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxin-HCl, 1.0mg/l thiamine-HCl, 1.0 g/l casamino acids, 20 g/l sucrose, and 2.0 g/lPhytogel, pH 5.8) on which they were kept for another 2 to 3 weeks,after which they were transferred to soil and cultivated in thegreenhouse.

The compositions of the 2N6, N67, N6M25 and hormone-free N6 media,described above, were kindly provided by Japan Tobacco Inc., PlantBreeding and Genetics Research Laboratory, 700 Higashibara, Toyoda,Iwata, Shizuoka 438, Japan.

EXAMPLE 10

Characterization of the Transformed Rice Plants of Example 9

Two transformed rice plants of Example 9, obtained in differenttransformation experiments, were cultivated for four weeks in soil andwere then sprayed with BASTA® at a dosage corresponding to 2 l/ha. Thetwo plants were BASTA^(R) resistant and survived the herbicide treatmentwhereas non-transformed control plants turned brown and died within fourdays of herbicide spraying.

The two plants and four other in vitro plantlets, derived from twofurther transformation experiments of Example 9, were analyzed by meansof a Southern hybridization in which plnat genomic DNA, digested withPvuII, was probed with pDE110. This analysis showed that, in allanalyzed plants, at least part of one copy of pDE110 was integrated inthe rice genome. In five out of six plants, the 1.6 kb fragmentcoresponding to the pDE110 fragment containing most of the 35S-barchimaeric gene could be unambiguously identified.

EXAMPLE 11

Field Tests with the Transformed Corn Plants of Examples 2 and 4

The progeny of the corn transformant H99-M148-1 of Example 2 and of thecorn transformant Pa91-M146-2 of Example 4 were tested under fieldconditions at the Plant Genetic Systems, N.V. experimental farm inAfsnee, Belgium. The field trial was authorized by the Belgian Ministryof Agriculture under registration number BIOT/91/M06. F1, F2, and F3progeny were obtained from crosses as summarized in Table 1, below. Inall cases one of the parents was assumed to be a heterozygote for theneo gene.

Up to 100 seeds of each seedlot were planted in 5 parellel rows, eachwith a length of 5 meters. Individual plants were 0.25 m apart, and thedistance between rows was 1 m. 10 rows of experimental plants werealternated with 1 row of non-transformed H99 and 1 row ofnon-transformed Pa91 plants as controls. One plot consisted of F1 and F2experimental plants with controls. Each of these plots was surrounded byi) a path with a width of 1 m and ii) 3 rows (1 m apart) ofnon-transformed corn plants (variety Sanora).

Experimental plots were prepared, sowed and maintained according to theschedule in Table 2, below. For sowing, plant holes were made with aplant stick, and seeds were put in by hand to a depth of 4 to 5 cm.

The field trial was terminated by manual removal and subsequent steamingof all cobs of the experimental plants. The remainders of the plantswere mechanically chopped with a mowing machine.

The following observations were made. At the 2-3 leaf stage, the totalnumber of germinated seeds was counted for each seedlot. As can be seenfrom Table 3, below, the percentage of germination varied between 63%and 100% with the exception of seedlot P4482, from which only 42% of theseeds germinated. Germination of seedlots of untransformed H99 and Pa91plants varied between 25% and 75%.

At the 3-4 leaf stage, the phenotype of the transgenic neo gene wasassayed as follows. In each plant, an incision up to the midvein wasmade in two leaves with a small pair of scissors. The incisions werethen brushed with a piece of cotton wool drenched in an aqueoussuspension of 4% kanamycin and 0.2% SDS. Some plants were treated with asuspension of 5% kanamycin and 0.1% SDS. A plant was scored as sensitiveand as lacking an active neo gene when the newly formed leaves wereyellow. A plant was scored as resistant and as having an active neo genewhen the newly formed leaves were normal and showed no bleaching.Discoloration of the newly formed leaves was assessed about 10 daysafter the brushing. 5-8% of the tested plants had an intermediatephenotype as they could not be unambiguously scored as sensitive orresistant. This was probably due to variations in environmentalconditions and/or developmental stages of the tested plants and a lessthan optimal kanamycin (and/or SDS) concentration.

In later analyses, the intermediate phenotypes were pooled with thesensitive plants. The proportions of kanamycin resistant plants versuskanamycin sensitive plants (including intermediate phenotypes) for eachcrossing or self was determined by a chi-square goodness of fit test(Snedecor and Cochran (1967) `Statistical Methods`, the Iowa StateUniversity Press, Ames, Iowa, U.S.A.) under the assumption of a onelocus Mendelian segregation of the neo gene. The results are summarizedin Table 3, below.

From the data in Table 3, it can be concluded that the introduced neogene remained stable over three generations regardless of whether theprogeny was obtained through selfing, backcrossing, or outcrossing to anunrelated line. The pattern of segregation was consistent with eachoriginal transformant having had only one active copy or multipleclosely linked active copies of the neo gene and with the neo gene traithaving had a normal Mendelian one-locus inheritance.

In all cases, the experimental plants appeared to be morphologicallycompletely normal when compared to untransformed control plants.

                  TABLE 1                                                         ______________________________________                                        Cross                     Seedlot                                             ______________________________________                                        F1      H99 × H99-M148-1                                                                              P3166                                                   Pa91 × H99-M148-1                                                                             P3169                                                   H99 × Pa91-M146-2                                                                             P3162                                                   Selfing of Pa91-M146-2                                                                              P3173 (1)                                       F2      P3169-024 × H99 P3651                                                   Selfing of P3166-002  P3989                                                   P3166-012 × H99 P3983                                                   P3166-018 × H99 P3982                                                   Selfing of P3173-003  P3996                                                   P3162-017 × H99 P4004                                                   P3162-008 × Pa91                                                                              P4008                                           F3      H99 × (P3166-005 × H99)-001                                                             P4481                                                   H99 × (P3162-004 × H99)-011                                                             P4483                                                   Selfing of(Selfing of P3166-001)-003                                                                P4482                                                   Pa91 × (P3169-028 × Pa91)-004                                                           P4310                                                   H99 × (P3169-036 × H99)-003                                                             P4306                                           ______________________________________                                         (1) not tested                                                           

                  TABLE 2                                                         ______________________________________                                        Date       Activity           Quantity                                        ______________________________________                                        March 29, 1991                                                                           lime treatment of soil                                                                           2000 kg/ha                                      May 23, 1991                                                                             NH.sub.4 NO.sub.3 treatment                                                                      740 kg/ha                                       May 23, 1991                                                                             superphosphate treatment                                                                         833 kg/ha                                       May 23, 1991                                                                             potassium sulphate 120 kg/ha                                       May 27, 1991                                                                             sowing of F1 and F2 seedlots                                                                     --                                              July 4, 1991                                                                             Herbicide treatment:                                                          Laddok             4 l/ha                                                     paraffin oil       105 l/ha                                        July 8, 1991                                                                             Sowing of F3 seedlots                                                                            --                                              July 26, 1991                                                                            Insecticide treatment:                                                        Pyrimor            0.265 kg/ha                                                Ambush             0.133 l/ha                                      October 10, 1991                                                                         termination        --                                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Code     Emerg   %     T   R   I   S   ND   X.sup.2                                                                            Sign.                        ______________________________________                                        F1  P3169    16/20   5   16   5  6   4   1    1.67 n.s.                           P3166    79/100  4   79  36  0   35  8    0.01 n.s.                           P3162    86/100  4   84  47  1   31  3    2.85 n.s.                       F2  P3651    65/100  4   62  26  11  16  9    0.02 n.s.                           P3989    91/100  4   83  66  1   10  5    4.71 p <0.05                        P3983    36/40   4   34  17  2   14  1    0.03 n.s.                           P3982    51/60   4   42  20  4   17  1    0.02 n.s.                           P3996    54/60   4   48  32  0   11  5    0.01 n.s.                           P4004    92/100  4   86  38  11  31  6    0.20 n.s.                           P4008    20/20   4   18   6  9   3   0    2.00 n.s.                       F3  P4481    72/100  5   66  32  2   30  2    0    n.s.                           P4483    63/100  5   47  22  2   23  0    0.19 n.s.                           P4482    42/100  5   34  30  0   4   0    3.18 n.s.                           P4310    84/100  5   82  50  7   24  1    4.46 p <0.05                        P4306    85/100  5   79  39  1   39  0    0.01 n.s.                       ______________________________________                                    

Code=seedlot (see Table 1); Emerg=number of seedlings per number ofsowed seeds; %=percentage of kanamycin in solution used in brushingassay; T=total number of plants tested; R=number of kanamycin resistantplants; I=number of intermediate phenotypes; S=number of kanamycinsensitive plants; ND=number of tested plants that were not scoredbecause seedlings were stopped in growth and died; X² =value ofchi-square for segregation of R versus I+S (expected values inoutcrossings are 50% R-50% I+S; expected values in selfings are 75%R-25% I+S under assumption of one locus segregation).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 5                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 5399 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: plasmid p - #DE108                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1..451                                                #/label= pUC18OTHER INFORMATION:                                              #"pUC18 derived sequence"                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 452..1284                                             #/label= 35S3 OTHER INFORMATION:                                              #"35S3 promoter sequence derived from                                         #mosaic virus isolate CabbB-JI"                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1285..2100                                            #/label= NPTIIOTHER INFORMATION:                                              #"coding sequence of neomycine                                                               phosphotrans - #ferase gene"                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 2101..3160                                            #/label= 3'ocsOTHER INFORMATION:                                              #"3' regulatory sequence containing the                                                      polyadenylat - #ion site derived from the                                     Agrobacteriu - #m T-DNA octopine synthase gene"                -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 3161..5399                                            #/label= pUC18OTHER INFORMATION:                                              #"pUC18 derived sequence"                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GA - #GACGGTCA         60                                                                          - CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TC - #AGCGGGTG        120                                                                          - TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CT - #GAGAGTGC        180                                                                          - ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC AT - #CAGGCGCC        240                                                                          - ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TC - #TTCGCTAT        300                                                                          - TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA AC - #GCCAGGGT        360                                                                          - TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT AC - #CCGGGGAT        420                                                                          - CCTCTAGAGT CGACCTGCAG GCATGCAAGC TCCTACGCAG CAGGTCTCAT CA - #AGACGATC        480                                                                          - TACCCGAGTA ACAATCTCCA GGAGATCAAA TACCTTCCCA AGAAGGTTAA AG - #ATGCAGTC        540                                                                          - AAAAGATTCA GGACTAATTG CATCAAGAAC ACAGAGAAAG ACATATTTCT CA - #AGATCAGA        600                                                                          - AGTACTATTC CAGTATGGAC GATTCAAGGC TTGCTTCATA AACCAAGGCA AG - #TAATAGAG        660                                                                          - ATTGGAGTCT CTAAAAAGGT AGTTCCTACT GAATCTAAGG CCATGCATGG AG - #TCTAAGAT        720                                                                          - TCAAATCGAG GATCTAACAG AACTCGCCGT GAAGACTGGC GAACAGTTCA TA - #CAGAGTCT        780                                                                          - TTTACGACTC AATGACAAGA AGAAAATCTT CGTCAACATG GTGGAGCACG AC - #ACTCTGGT        840                                                                          - CTACTCCAAA AATGTCAAAG ATACAGTCTC AGAAGACCAA AGGGCTATTG AG - #ACTTTTCA        900                                                                          - ACAAAGGATA ATTTCGGGAA ACCTCCTCGG ATTCCATTGC CCAGCTATCT GT - #CACTTCAT        960                                                                          - CGAAAGGACA GTAGAAAAGG AAGGTGGCTC CTACAAATGC CATCATTGCG AT - #AAAGGAAA       1020                                                                          - GGCTATCATT CAAGATGCCT CTGCCGACAG TGGTCCCAAA GATGGACCCC CA - #CCCACGAG       1080                                                                          - GAGCATCGTG GAAAAAGAAG ACGTTCCAAC CACGTCTTCA AAGCAAGTGG AT - #TGATGTGA       1140                                                                          - CATCTCCACT GACGTAAGGG ATGACGCACA ATCCCACTAT CCTTCGCAAG AC - #CCTTCCTC       1200                                                                          - TATATAAGGA AGTTCATTTC ATTTGGAGAG GACACGCTGA AATCACCAGT CT - #CTCTCTAT       1260                                                                          - AAATCTATCT CTCTCTCTAT AACCATGGAT CCGGCCAAGC TAGCTTGGAT TG - #AACAAGAT       1320                                                                          - GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TG - #ACTGGGCA       1380                                                                          - CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GG - #GGCGCCCG       1440                                                                          - GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAGGA CG - #AGGCAGCG       1500                                                                          - CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CG - #TTGTCACT       1560                                                                          - GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CC - #TGTCATCT       1620                                                                          - CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GC - #TGCATACG       1680                                                                          - CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GC - #GAGCACGT       1740                                                                          - ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TC - #AGGGGCTC       1800                                                                          - GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGCGCATGC CCGACGGCGA GG - #ATCTCGTC       1860                                                                          - GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CT - #TTTCTGGA       1920                                                                          - TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GT - #TGGCTACC       1980                                                                          - CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GC - #TTTACGGT       2040                                                                          - ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GT - #TCTTCTGA       2100                                                                          - GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TC - #ACGAGATT       2160                                                                          - TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CG - #GGACGCCG       2220                                                                          - GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CC - #CCTGCTTT       2280                                                                          - AATGAGATAT GCGAGACGCC TATGATCGCA TGATATTTGC TTTCAATTCT GT - #TGTGCACG       2340                                                                          - TTGTAAAAAA CCTGAGCATG TGTAGCTCAG ATCCTTACCG CCGGTTTCGG TT - #CATTCTAA       2400                                                                          - TGAATATATC ACCCGTTACT ATCGTATTTT TATGAATAAT ATTCTCCGTT CA - #ATTTACTG       2460                                                                          - ATTGTACCCT ACTACTTATA TGTACAATAT TAAAATGAAA ACAATATATT GT - #GCTGAATA       2520                                                                          - GGTTTATAGC GACATCTATG ATAGAGCGCC ACAATAACAA ACAATTGCGT TT - #TATTATTA       2580                                                                          - CAAATCCAAT TTTAAAAAAA GCGGCAGAAC CGGTCAAACC TAAAAGACTG AT - #TACATAAA       2640                                                                          - TCTTATTCAA ATTTCAAAAG GCCCCAGGGG CTAGTATCTA CGACACACCG AG - #CGGCGAAC       2700                                                                          - TAATAACGTT CACTGAAGGG AACTCCGGTT CCCCGCCGGC GCGCATGGGT GA - #GATTCCTT       2760                                                                          - GAAGTTGAGT ATTGGCCGTC CGCTCTACCG AAAGTTACGG GCACCATTCA AC - #CCGGTCCA       2820                                                                          - GCACGGCGGC CGGGTAACCG ACTTGCTGCC CCGAGAATTA TGCAGCATTT TT - #TTGGTGTA       2880                                                                          - TGTGGGCCCC AAATGAAGTG CAGGTCAAAC CTTGACAGTG ACGACAAATC GT - #TGGGCGGG       2940                                                                          - TCCAGGGCGA ATTTTGCGAC AACATGTCGA GGCTCAGCAG GGGCTCGATC CC - #CTCGCGAG       3000                                                                          - TTGGTTCAGC TGCTGCCTGA GGCTGGACGA CCTCGCGGAG TTCTACCGGC AG - #TGCAAATC       3060                                                                          - CGTCGGCATC CAGGAAACCA GCAGCGGCTA TCCGCGCATC CATGCCCCCG AA - #CTGCAGGA       3120                                                                          - GTGGGGAGGC ACGATGGCCG CTTTGGTCGA CCTGCAGCCA AGCTTGGCGT AA - #TCATGGTC       3180                                                                          - ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TA - #CGAGCCGG       3240                                                                          - AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC TAACTCACAT TA - #ATTGCGTT       3300                                                                          - GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC CAGCTGCATT AA - #TGAATCGG       3360                                                                          - CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CG - #CTCACTGA       3420                                                                          - CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AG - #GCGGTAAT       3480                                                                          - ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AA - #GGCCAGCA       3540                                                                          - AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TC - #CGCCCCCC       3600                                                                          - TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CA - #GGACTATA       3660                                                                          - AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CG - #ACCCTGCC       3720                                                                          - GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CT - #CAATGCTC       3780                                                                          - ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GT - #GTGCACGA       3840                                                                          - ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AG - #TCCAACCC       3900                                                                          - GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GC - #AGAGCGAG       3960                                                                          - GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT AC - #ACTAGAAG       4020                                                                          - GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GA - #GTTGGTAG       4080                                                                          - CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GC - #AAGCAGCA       4140                                                                          - GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CG - #GGGTCTGA       4200                                                                          - CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CA - #AAAAGGAT       4260                                                                          - CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GT - #ATATATGA       4320                                                                          - GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CA - #GCGATCTG       4380                                                                          - TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CG - #ATACGGGA       4440                                                                          - GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CA - #CCGGCTCC       4500                                                                          - AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GT - #CCTGCAAC       4560                                                                          - TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GT - #AGTTCGCC       4620                                                                          - AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CA - #CGCTCGTC       4680                                                                          - GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CA - #TGATCCCC       4740                                                                          - CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GA - #AGTAAGTT       4800                                                                          - GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CT - #GTCATGCC       4860                                                                          - ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GA - #GAATAGTG       4920                                                                          - TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CG - #CCACATAG       4980                                                                          - CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TC - #TCAAGGAT       5040                                                                          - CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT GA - #TCTTCAGC       5100                                                                          - ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA AT - #GCCGCAAA       5160                                                                          - AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TT - #CAATATTA       5220                                                                          - TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GT - #ATTTAGAA       5280                                                                          - AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG AC - #GTCTAAGA       5340                                                                          - AACCATTATT ATCATGACAT TAACCTATAA AAATAGGCGT ATCACGAGGC CC - #TTTCGTC        5399                                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1186 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: probe                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1..8                                                  #/note= "sequence derived fromN:                                                             tapetum s - #pecific promoter of Nicotiana tabacum"            -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 9..790                                                #/label= NPTIIOTHER INFORMATION:                                              #"coding sequence of neomycine                                                               phosphotrans - #ferase gene"                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 791..1186                                             #/label= 3'g7 OTHER INFORMATION:                                              #"3' regulatory sequence containing the                                                      polyadenylat - #ion site derived from Agrobacterium                           T-DNA gen - #e 7"                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - AAGCTTGGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TA - #TTCGGCTA         60                                                                          - TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TG - #TCAGCGCA        120                                                                          - GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AA - #CTGCAGGA        180                                                                          - CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CT - #GTGCTCGA        240                                                                          - CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GG - #CAGGATCT        300                                                                          - CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CA - #ATGCGGCG        360                                                                          - GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC AT - #CGCATCGA        420                                                                          - GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG AC - #GAAGAGCA        480                                                                          - TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGCGCATGC CC - #GACGGCGA        540                                                                          - GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AA - #AATGGCCG        600                                                                          - CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AG - #GACATAGC        660                                                                          - GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GC - #TTCCTCGT        720                                                                          - GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TT - #CTTGACGA        780                                                                          - GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CA - #ACCTGCCA        840                                                                          - TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AA - #TCGTTTTC        900                                                                          - CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CT - #TCGCCCAC        960                                                                          - CCCGATCCAT GAGCTAAGCT AGCTATATCA TCAATTTATG TATTACACAT AA - #TATCGCAC       1020                                                                          - TCAGTCTTTC ATCTACGGCA ATGTACCAGC TGATATAATC AGTTATTGAA AT - #ATTTCTGA       1080                                                                          - ATTTAAACTT GCATCAATAA ATTTATGTTT TTGCTTGGAC TATAATACCT GA - #CTTGTTAT       1140                                                                          #               1186AAA CTATATTTCT TTCAAGATGG GAATTC                          - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1287 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #of pTTM8 (A) ORGANISM: fragment                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1..545                                                #/label= PTA29OTHER INFORMATION:                                              #"Promoter from the TA29 gene of Nicotiana                                                   tabacum"                                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 546..881                                              #/label= barnaseHER INFORMATION:                                              #"coding sequence of barnase gene"                                            -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 882..1287                                             #/label= 3'nosOTHER INFORMATION:                                              #"3' regulatory sequence containing the                                                      polyadenylat - #ion site derived from Agrobacterium                           T-DNA nop - #aline synthase gene "                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATCTAGCTAA GTATAACTGG ATAATTTGCA TTAACAGATT GAATATAGTG CC - #AAACAAGA         60                                                                          - AGGGACAATT GACTTGTCAC TTTATGAAAG ATGATTCAAA CATGATTTTT TA - #TGTACTAA        120                                                                          - TATATACATC CTACTCGAAT TAAAGCGACA TAGGCTCGAA GTATGCACAT TT - #AGCAATGT        180                                                                          - AAATTAAATC AGTTTTTGAA TCAAGCTAAA AGCAGACTTG CATAAGGTGG GT - #GGCTGGAC        240                                                                          - TAGAATAAAC ATCTTCTCTA GCACAGCTTC ATAATGTAAT TTCCATAACT GA - #AATCAGGG        300                                                                          - TGAGACAAAA TTTTGGTACT TTTTCCTCAC ACTAAGTCCA TGTTTGCAAC AA - #ATTAATAC        360                                                                          - ATGAAACCTT AATGTTACCC TCAGATTAGC CTGCTACTCC CCATTTTCCT CG - #AAATGCTC        420                                                                          - CAACAAAAGT TAGTTTTGCA AGTTGTTGTG TATGTCTTGT GCTCTATATA TG - #CCCTTGTG        480                                                                          - GTGCAAGTGT AACAGTACAA CATCATCACT CAAATCAAAG TTTTTACTTA AA - #GAAATTAG        540                                                                          - CTACCATGGT ACCGGTTATC AACACGTTTG ACGGGGTTGC GGATTATCTT CA - #GACATATC        600                                                                          - ATAAGCTACC TGATAATTAC ATTACAAAAT CAGAAGCACA AGCCCTCGGC TG - #GGTGGCAT        660                                                                          - CAAAAGGGAA CCTTGCAGAC GTCGCTCCGG GGAAAAGCAT CGGCGGAGAC AT - #CTTCTCAA        720                                                                          - ACAGGGAAGG CAAACTCCCG GGCAAAAGCG GACGAACATG GCGTGAAGCG GA - #TATTAACT        780                                                                          - ATACATCAGG CTTCAGAAAT TCAGACCGGA TTCTTTACTC AAGCGACTGG CT - #GATTTACA        840                                                                          - AAACAACGGA CCATTATCAG ACCTTTACAA AAATCAGATA ACGAAAAAAA CG - #GCTTCCTG        900                                                                          - CGGAGGCCGT TTTTTTCAGC TTTACATAAA GTGTGTAATA AATTTTTCTT CA - #AACTCTGA        960                                                                          - TCGGTCAATT TCACTTTCCG GNNNNCTCTA GAGGATCCGA AGCAGATCGT TC - #AAACATTT       1020                                                                          - GGCAATAAAG TTTCTTAAGA TTGAATCCTG TTGCCGGTCT TGCGATGATT AT - #CATATAAT       1080                                                                          - TTCTGTTGAA TTACGTTAAG CATGTAATAA TTAACATGTA ATGCATGACG TT - #ATTTATGA       1140                                                                          - GATGGGTTTT TATGATTAGA GTCCCGCAAT TATACATTTA ATACGCGATA GA - #AAACAAAA       1200                                                                          - TATAGCGCGC AAACTAGGAT AAATTATCGC GCGCGGTGTC ATCTATGTTA CT - #AGATCGGG       1260                                                                          #           1287   GAGC TCGAATT                                               - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 4883 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: plasmid p - #DE110                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1..395                                                #/label= pUC18OTHER INFORMATION:                                              #"pUC18 derived sequence"                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 396..1779                                             #/label= 35S3 OTHER INFORMATION:                                              #"35S3 promoter sequence derived from                                         #mosaic virus isolate CabbB-JI"                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 1780..2331                                            #/label= bar) OTHER INFORMATION:                                              #"coding sequence of phosphinothricin                                                        acetyltransf - #erase gene"                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 2332..2619                                            #/label= 3'nosOTHER INFORMATION:                                              #"3' regulatory sequence containing the                                                      polyadenylat - #ion site derived from Agrobacterium                           T-DNA nop - #aline synthase gene"                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                                 (B) LOCATION: 2620..4883                                            #/label= pUC18OTHER INFORMATION:                                              #"pUC18 derived sequence"                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GA - #GACGGTCA         60                                                                          - CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TC - #AGCGGGTG        120                                                                          - TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CT - #GAGAGTGC        180                                                                          - ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC AT - #CAGGCGCC        240                                                                          - ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TC - #TTCGCTAT        300                                                                          - TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA AC - #GCCAGGGT        360                                                                          - TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CCAATCCCAC CA - #AAACCTGA        420                                                                          - ACCTAGCAGT TCAGTTGCTC CTCTCAGAGA CGAATCGGGT ATTCAACACC CT - #CATACCAA        480                                                                          - CTACTACGTC GTGTATAACG GACCTCATGC CGGTATATAC GATGACTGGG GT - #TGTACAAA        540                                                                          - GGCAGCAACA AACGGTGTTC CCGGAGTTGC GCATAAGAAG TTTGCCACTA TT - #ACAGAGGC        600                                                                          - AAGAGCAGCA GCTGACGCGT ATACAACAAG TCAGCAAACA GATAGGTTGA AC - #TTCATCCC        660                                                                          - CAAAGGAGAA GCTCAACTCA AGCCCAAGAG CTTTGCGAAG GCCCTAACAA GC - #CCACCAAA        720                                                                          - GCAAAAAGCC CACTGCTCAC GCTAGGAACC AAAAGGCCCA GCAGTGATCC AG - #CCCCAAAA        780                                                                          - GAGATCTCCT TTGCCCCGGA GATTACAATG GACGATTTCC TCTATCTTTA CG - #ATCTAGGA        840                                                                          - AGGAAGTTCG AAGGTGAAGG TGACGACACT ATGTTCACCA CTGATAATGA GA - #AGGTTAGC        900                                                                          - CTCTTCAATT TCAGAAAGAA TGCTGACCCA CAGATGGTTA GAGAGGCCTA CG - #CAGCAGGT        960                                                                          - CTCATCAAGA CGATCTACCC GAGTAACAAT CTCCAGGAGA TCAAATACCT TC - #CCAAGAAG       1020                                                                          - GTTAAAGATG CAGTCAAAAG ATTCAGGACT AATTGCATCA AGAACACAGA GA - #AAGACATA       1080                                                                          - TTTCTCAAGA TCAGAAGTAC TATTCCAGTA TGGACGATTC AAGGCTTGCT TC - #ATAAACCA       1140                                                                          - AGGCAAGTAA TAGAGATTGG AGTCTCTAAA AAGGTAGTTC CTACTGAATC TA - #AGGCCATG       1200                                                                          - CATGGAGTCT AAGATTCAAA TCGAGGATCT AACAGAACTC GCCGTGAAGA CT - #GGCGAACA       1260                                                                          - GTTCATACAG AGTCTTTTAC GACTCAATGA CAAGAAGAAA ATCTTCGTCA AC - #ATGGTGGA       1320                                                                          - GCACGACACT CTGGTCTACT CCAAAAATGT CAAAGATACA GTCTCAGAAG AC - #CAAAGGGC       1380                                                                          - TATTGAGACT TTTCAACAAA GGATAATTTC GGGAAACCTC CTCGGATTCC AT - #TGCCCAGC       1440                                                                          - TATCTGTCAC TTCATCGAAA GGACAGTAGA AAAGGAAGGT GGCTCCTACA AA - #TGCCATCA       1500                                                                          - TTGCGATAAA GGAAAGGCTA TCATTCAAGA TGCCTCTGCC GACAGTGGTC CC - #AAAGATGG       1560                                                                          - ACCCCCACCC ACGAGGAGCA TCGTGGAAAA AGAAGACGTT CCAACCACGT CT - #TCAAAGCA       1620                                                                          - AGTGGATTGA TGTGACATCT CCACTGACGT AAGGGATGAC GCACAATCCC AC - #TATCCTTC       1680                                                                          - GCAAGACCCT TCCTCTATAT AAGGAAGTTC ATTTCATTTG GAGAGGACAC GC - #TGAAATCA       1740                                                                          - CCAGTCTCTC TCTATAAATC TATCTCTCTC TCTATAACCA TGGACCCAGA AC - #GACGCCCG       1800                                                                          - GCCGACATCC GCCGTGCCAC CGAGGCGGAC ATGCCGGCGG TCTGCACCAT CG - #TCAACCAC       1860                                                                          - TACATCGAGA CAAGCACGGT CAACTTCCGT ACCGAGCCGC AGGAACCGCA GG - #AGTGGACG       1920                                                                          - GACGACCTCG TCCGTCTGCG GGAGCGCTAT CCCTGGCTCG TCGCCGAGGT GG - #ACGGCGAG       1980                                                                          - GTCGCCGGCA TCGCCTACGC GGGCCCCTGG AAGGCACGCA ACGCCTACGA CT - #GGACGGCC       2040                                                                          - GAGTCGACCG TGTACGTCTC CCCCCGCCAC CAGCGGACGG GACTGGGCTC CA - #CGCTCTAC       2100                                                                          - ACCCACCTGC TGAAGTCCCT GGAGGCACAG GGCTTCAAGA GCGTGGTCGC TG - #TCATCGGG       2160                                                                          - CTGCCCAACG ACCCGAGCGT GCGCATGCAC GAGGCGCTCG GATATGCCCC CC - #GCGGCATG       2220                                                                          - CTGCGGGCGG CCGGCTTCAA GCACGGGAAC TGGCATGACG TGGGTTTCTG GC - #AGCTGGAC       2280                                                                          - TTCAGCCTGC CGGTACCGCC CCGTCCGGTC CTGCCCGTCA CCGAGATCTG AT - #CTCACGCG       2340                                                                          - TCTAGGATCC GAAGCAGATC GTTCAAACAT TTGGCAATAA AGTTTCTTAA GA - #TTGAATCC       2400                                                                          - TGTTGCCGGT CTTGCGATGA TTATCATATA ATTTCTGTTG AATTACGTTA AG - #CATGTAAT       2460                                                                          - AATTAACATG TAATGCATGA CGTTATTTAT GAGATGGGTT TTTATGATTA GA - #GTCCCGCA       2520                                                                          - ATTATACATT TAATACGCGA TAGAAAACAA AATATAGCGC GCAAACTAGG AT - #AAATTATC       2580                                                                          - GCGCGCGGTG TCATCTATGT TACTAGATCG GGAAGATCCT CTAGAGTCGA CC - #TGCAGGCA       2640                                                                          - TGCAAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT AT - #CCGCTCAC       2700                                                                          - AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CC - #TAATGAGT       2760                                                                          - GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GA - #AACCTGTC       2820                                                                          - GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GT - #ATTGGGCG       2880                                                                          - CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GG - #CGAGCGGT       2940                                                                          - ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA AC - #GCAGGAAA       3000                                                                          - GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CG - #TTGCTGGC       3060                                                                          - GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CA - #AGTCAGAG       3120                                                                          - GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GC - #TCCCTCGT       3180                                                                          - GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TC - #CCTTCGGG       3240                                                                          - AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AG - #GTCGTTCG       3300                                                                          - CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CC - #TTATCCGG       3360                                                                          - TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CA - #GCAGCCAC       3420                                                                          - TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TG - #AAGTGGTG       3480                                                                          - GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TG - #AAGCCAGT       3540                                                                          - TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CT - #GGTAGCGG       3600                                                                          - TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AA - #GAAGATCC       3660                                                                          - TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AA - #GGGATTTT       3720                                                                          - GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AA - #TGAAGTTT       3780                                                                          - TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GC - #TTAATCAG       3840                                                                          - TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GA - #CTCCCCGT       3900                                                                          - CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CA - #ATGATACC       3960                                                                          - GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CC - #GGAAGGGC       4020                                                                          - CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA AT - #TGTTGCCG       4080                                                                          - GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CC - #ATTGCTAC       4140                                                                          - AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GT - #TCCCAACG       4200                                                                          - ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CC - #TTCGGTCC       4260                                                                          - TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TG - #GCAGCACT       4320                                                                          - GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GT - #GAGTACTC       4380                                                                          - AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CG - #GCGTCAAT       4440                                                                          - ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GA - #AAACGTTC       4500                                                                          - TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TG - #TAACCCAC       4560                                                                          - TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GG - #TGAGCAAA       4620                                                                          - AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GT - #TGAATACT       4680                                                                          - CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TC - #ATGAGCGG       4740                                                                          - ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CA - #TTTCCCCG       4800                                                                          - AAAAGTGCCA CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT AT - #AAAAATAG       4860                                                                          #              4883TTTC GTC                                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #        10                                                                   __________________________________________________________________________

We claim:
 1. A process for the stable integration of a DNA, comprising agene that is functional in a cell of a cereal plant, wherein said DNA isintegrated into the nuclear genome of said cereal plant, said processcomprising the steps of:(a) providing a compact embryogenic callus ofsaid cereal plant; (b) wounding said compact embryogenic callus ortreating said compact embryogenic callus with a cell wall degradingenzyme for a period of time so as not to cause a complete disruption oftissues, and transferring said DNA into the nuclear genome of a cell insaid compact embryogenic callus by means of Agrobacterium-mediatedtransformation to generate a transformed cell; and (c) regenerating atransformed cereal plant from said transformed cell.
 2. The process ofclaim 1, wherein said compact embryogenic callus is wounded by cuttingto callus pieces.
 3. The process of claim 2, wherein said callus pieceshave a maximum length of 0.5 to 2.5 mm.
 4. The process of claim 3,wherein said callus pieces have a maximum length of 1 to 2 mm.
 5. Theprocess of claim 1, wherein said compact embryogenic callus is treatedwith said enzyme for a period of time so as not to cause a completedisruption of tissues.
 6. The process of claim 5, wherein said compactembryogenic callus is treated with an enzyme that degrades the cellwalls of said callus for a period of 1 to 10 minutes.
 7. The process ofclaim 6, wherein said callus is treated with said enzyme for a period of1 to 2 minutes.
 8. The process of claim 1, wherein said compactembryogenic callus is subjected to a period of plasmolysis prior totransformation.
 9. The process of claim 1, wherein said gene comprisesthe DNA encoding barnase under control of a stamen specific promoter ofthe TA29 gene of tobacco with a sequence within the DNA of SEQ ID No. 3between nucleotide 1 and
 545. 10. The process of claim 1, wherein saidgene comprises a DNA coding for a phosphinotricin acetyltransferase or aneomycin phosphotransferase.
 11. The process of claim 1, wherein saidcereal plant is selected from the group consisting of corn, wheat, riceand rye.
 12. The process of claim 1, wherein said cereal plant is wheat.13. The process of claim 10, wherein said cereal plant is wheat.
 14. Theprocess of claim 1, wherein said compact embryogenic callus ispropagated from scutella of embryos.